JP7710993B2 - Homopolymer-encoded nucleic acid memory - Google Patents

Description

RELATED APPLICATIONS This application claims priority to U.S. patent application Ser. No. 16/393,510, filed Apr. 24, 2019, the contents of which are incorporated herein by reference.

FIELD OF THEINVENTION The present invention relates to methods and devices for storing data in nucleic acid memory strands that contain homopolymer tracts.

Background DNA digital storage is a process that uses base sequences in DNA to represent digital data and store that data via DNA synthesis of polynucleotides that correspond to the base sequences that encode the data. DNA digital storage offers several advantages over traditional data storage methods and targets a multi-billion dollar market. Traditional data storage methods, including flash memory and recording on magnetic tape, pose physical space requirements, reliance on scarce resources, and problems with data integrity. DNA digital storage offers significantly greater data storage density with significantly lower energy requirements. Current methods rely on high-fidelity sequencing techniques with little tolerance for errors to accurately read the data encoded in DNA. The required sequencing methods are relatively slow and expensive to meet the fidelity requirements. One example of a current DNA digital storage technique is described in U.S. Patent No. 9,384,320 to Church, et al. (incorporated herein by reference). To increase the fidelity of sequencing, current methods such as those described by Church encode data using sequences that avoid features that are difficult to read or write, such as sequence repeats.

The synthesis side of current DNA digital storage techniques further limits the adoption of this technology through lack of speed, production of toxic by-products and high cost. Most de novo nucleic acid sequences are synthesized using solid-phase phosphoramidite techniques that involve sequential deprotection and synthesis of sequences constructed from phosphoramidite reagents corresponding to natural (or unnatural) nucleic acid bases. Inkjet synthesis on array-based formats allows for very low-cost phosphoramidite synthesis, but the strands produced are limited to 100-200 bases in length, must sacrifice some length to index the sequence, and are produced at subfemtomolar scales that require post-synthesis amplification to provide sufficient material for subsequent readout. Using conventional synthesis techniques, nucleic acids longer than 200 base pairs (bp) in length undergo high rates of destruction and side reactions. Furthermore, conventional synthesis techniques produce toxic by-products, and disposal of this waste limits the availability of nucleic acid synthesizers and increases the cost of oligo production. These formidable problems associated with synthesis and readout of DNA digital storage have limited applications for an otherwise promising technology.

U.S. Pat. No. 9,384,320

SUMMARY The present invention provides systems and methods for storing data using sequences of homopolymer tracts that encode digital data. Using homopolymer tracts of repeated bases (e.g., 2-10 nucleotides) to represent each bit in the data sequence allows higher throughput and less expensive sequencing techniques to be used. Because sequence reading relies only on distinguishing transitions between homopolymer tracts and does not require reliable reading of each individual nucleotide, sequencing techniques such as nanopore sequencing, zero mode waveguide (ZMW) single molecule sequencing and mass spectrometry can be used to increase speed and reduce cost.

Recording of data using the homopolymer tracts described herein is most efficiently accomplished using long strands (e.g., 5-10 kb) of nucleic acid. While traditional synthesis techniques are length-limited, template-independent polynucleotide synthesis using, for example, nucleotidyl transferases, is capable of synthesizing long strands at reduced cost and with less waste product production. Enzymatically synthesized ssDNA memory strands require only 50% of the DNA synthesis compared to the traditional phosphoramidite approach, because ssDNA strands longer than about 100-200 nucleotides in length require complex and costly ligation or PCR techniques and can only produce ssDNA from dsDNA intermediates. See U.S. Patent No. 8,808,989 to Efcavitch, et al., incorporated herein by reference. Data encoding can be numeric base 2, base 3, base 4 using standard nucleotides, or data density can be increased using any number of modified nucleotide analogs to generate base 8, base 10, base 12 or higher encoding schemes.

The only limitations on the modified nucleotide analogs are that they can be incorporated using a selected synthetic technique (e.g., terminal deoxynucleotidyl transferase (TdT)) and can be distinguished from one another using a selected sequencing analysis. In some embodiments, synthesis can be accomplished using polymerase theta in the presence of Mn2 ⁺ .

Consistent homopolymer tract length is not essential to the systems and methods of the invention since only the transitions between individual tracts need to be recognized. Although tract length may be allowed to vary, the synthesis techniques of the invention effectively control the average homopolymer tract length by adjusting the ratio of deoxynucleotides (dNTPs) to the oligonucleotide memory strand being synthesized and controlling the exposure time of dNTPs to the nascent memory strand. The length of the homopolymer tract can be optimized for the readout technology; the highest data storage density is achieved with single nucleotide readout resolution, while the highest readout speed and accuracy is achieved by extending the size of the nucleotide bit to the minimum detectable length (e.g., 2-10 nucleotides) for a given sequencing technology.

Systems and methods of the invention using nanopore sequencing may use specialized memory strand constructs, such as stoppers (e.g., hairpins or polymeric appendages) included on one or both ends of the strand. In other nanopore-based methods, the memory strand may be circularized and threaded between adjacent nanopores.

Certain aspects of the invention include a method of recording data using a nucleic acid memory strand. The steps of the method may include creating an in-silico oligonucleotide sequence representing a data set, where each nucleotide of the oligonucleotide sequence corresponds to a unit of the data set. A nucleic acid memory strand may then be synthesized that includes a plurality of homopolymer tracts, where each homopolymer tract corresponds to a nucleotide of the oligonucleotide sequence. The plurality of homopolymer tracts may include between 3 and 10 repeated nucleotides. Each unit of the data set may be represented by a base 2, base 3, base 4 or higher base, as desired for a particular application.

In certain embodiments, the nucleic acid memory strand can be at least about 200 nucleotides long to about 5,000 nucleotides long. The synthesizing step can include controlling the homopolymer length by varying the dNTP concentration. The steps of the method can include modifying a first end of the nucleic acid memory strand to prevent passage of the first end through a nanopore of a nanopore sequencing system; passing a second end of the nucleic acid memory strand through the nanopore; and modifying a second end of the nucleic acid memory strand to prevent passage of the second end through the nanopore.

Other embodiments may use memory strands composed of heteropolymer tracts of defined stoichiometry or composition to further increase the coding capacity of a set number of nucleotide analogs. Further embodiments may aim to protect the data encoded in the memory strands by using nucleotide analogs that are similar in structure but employ linkers that can be removed under different conditions, e.g., ultraviolet or visible light, oxidizing or reducing agents, alkaline or acidic pH, or sequence-specific nucleases, thereby concealing the data to them without knowledge of the applicable process.

The data set may be selected from the group consisting of a text file, an image file, and an audio file. The synthesizing step may include template-independent synthesis. In certain embodiments, a nucleotidyl transferase enzyme may be used to catalyze the template-independent synthesis. In some embodiments, polymerase theta may be used to catalyze the template-independent synthesis.

Aspects of the invention may include a method of reading data from a nucleic acid memory strand. The steps of the method may include sequencing a nucleic acid memory strand including a plurality of homopolymer tracts, converting the nucleic acid memory strand sequence into digitized data, each of the plurality of homopolymer tracts representing nucleotides corresponding to a unit of data, and converting the digitized piece of data into a readable format. The steps of the method may include displaying the readable format. The plurality of homopolymer tracts may include repeats of between about 2 and about 10 nucleotides. The nucleic acid memory strand may be between about 200 and about 5,000 nucleotides in length.

In various embodiments, the sequencing step may include nanopore sequencing, sequencing by synthesis, or mass spectrometry. The sequencing, converting, and converting steps may be repeated one or more times on the nucleic acid memory strand.

In certain embodiments, for example, the following items are provided:
(Item 1)
1. A method for synthesizing a plurality of nucleic acid memory strands, comprising:
providing an array of two or more substrate-linked nucleic acids with addressable delivery of activation energy to each of said substrate-linked nucleic acids;
extending one or more of the substrate-linked nucleic acids with a homopolymeric tract of two or more repeating nucleotides by delivering an addressable activation energy to one or more of the substrate-linked nucleic acids in the presence of a plurality of blocked nucleotide analogs and a template-free polymerase, wherein the template-free polymerase incorporates unblocked nucleotide analogs but not blocked nucleotide analogs, and the addressable activation energy converts the blocked nucleotide analogs to unblocked nucleotide analogs;
The method includes:
(Item 2)
2. The method of claim 1, wherein the blocked nucleotide analog is converted to an unblocked nucleotide analog by removal of the blocking group.
(Item 3)
2. The method of claim 1, wherein the addressable activation energy comprises light, reducing conditions, a pH change, or heat.
(Item 4)
3. The method of claim 2, wherein the removable blocking group is at the 3'-OH of the blocked nucleotide analog.
(Item 5)
3. The method of claim 2, wherein the removable blocking group is on a purine or pyrimidine base of the nucleotide analogue.
(Item 6)
2. The method of claim 1, wherein the blocked nucleotide analog comprises a removable blocking group on the 3'-OH of the deoxyribose or ribose of a nucleotide triphosphate and a non-removable modification on a purine or pyrimidine base of the nucleotide analog.
(Item 7)
2. The method of claim 1, wherein the multiple blocked nucleotide analogs are modified nucleotides of the same nucleobase comprising a removable 3'-O-blocking group and two or more non-removable molecular modifications that allow for discrimination between modified nucleotide analogs of the same nucleobase.
(Item 8)
stopping the elongation;
extending the homopolymer tract with an additional homopolymer tract of two or more repeating nucleotides by delivering an addressable activation energy to the homopolymer tract in the presence of another plurality of blocked nucleotide analogs and the template-independent polymerase;
2. The method of claim 1, further comprising:
(Item 9)
9. The method of claim 8, wherein the extension is stopped after a predetermined amount of time to obtain a desired length for the homopolymer tract.
(Item 10)
2. The method of claim 1, wherein the rate of extension is regulated by modifications to the blocked nucleotide analog.
(Item 11)
11. The method of claim 10, wherein the rate controlling modifications are removed from the homopolymer tract after elongation.
(Item 12)
2. The method of claim 1, wherein the rate-regulating modification is removed during elongation.
(Item 13)
2. The method of claim 1, wherein the repeating nucleotides of the homopolymer tract are between 2 and about 10.
(Item 14)
9. The method of claim 8, further comprising repeating the terminating and extending steps to synthesize a nucleic acid memory strand.
(Item 15)
15. The method of claim 14, wherein the nucleic acid memory strand is about 200 nucleotides to about 5,000 nucleotides in length.
(Item 16)
2. The method of claim 1, wherein a predetermined concentration of the blocked nucleotide analog is provided in the extending step to obtain a desired length for the homopolymer tract.
(Item 17)
9. The method of claim 8, wherein the homopolymer tract and the further homopolymer tract comprise different nucleobases.
(Item 18)
15. The method of claim 14, wherein the nucleic acid memory strand encodes a data set selected from the group consisting of a text file, an image file, and an audio file.
(Item 19)
20. The method of claim 18, further comprising the step of displaying a readable format of the data set.
(Item 20)
Item 15. The method according to item 14, wherein the unit of data is expressed in base 2.
(Item 21)
Item 15. The method according to item 14, wherein the unit of data is expressed in base 3.
(Item 22)
Item 15. The method according to item 14, wherein the unit of data is expressed in base 4.
(Item 23)
15. The method according to claim 14, wherein the units of data are represented in a base greater than base 4.
(Item 24)
15. The method of claim 14, wherein the data is represented by the degree of decaging or the resulting tract length at individual steps of memory chain synthesis.
(Item 25)
15. The method of claim 14, wherein the data encoded in the nucleic acid memory strand is read by DNA sequencing.
(Item 26)
15. The method of claim 14, wherein the data encoded in the nucleic acid memory strand is read by passage of the nucleic acid memory strand through a nanopore.
Other aspects of the invention will be apparent to those skilled in the art in view of the following figures and detailed description.

FIG. 1 shows the enzymatic synthesis cycle used to form homopolymer tracts.

FIG. 2 shows a method for synthesizing a nucleic acid memory strand having a homopolymer tract.

FIG. 3 illustrates a method for reading data from a nucleic acid memory strand having a homopolymer tract.

FIG. 4 shows the relationship between chains/GB and homopolymer length and chain length.

FIG. 5 shows the data that can be encoded in a DNA strand composed of 500 polymer tracts as a function of the number of distinguishable nucleotide analogs in the memory strand.

FIG. 6 shows a nanopore trapped nucleic acid memory strand of the present invention.

FIG. 7 shows a scheme for varying the data encoded within a chain of homopolymer tracts in response to processing conditions.

FIG. 8 shows a system for synthesizing nucleic acid memory strands having homopolymer tracts.

FIG. 9 shows a system for parallel synthesis of nucleic acid memory strands having homopolymer tracts on an array of nanowells.

FIG. 10 provides a more detailed schematic diagram of components that may appear in the system.

FIG. 11 shows an analysis of the enzymatically mediated synthesis of homopolymer tracts of two different base compositions.

FIG. 12 shows the process for converting character text to a 12-bit nucleic acid sequence.

FIG. 13 shows polyacrylamide gel electrophoresis (PAGE) analysis of 12 cycles of enzymatic synthesis.

14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bits of homopolymer addition. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bits of homopolymer addition. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bit homopolymer additions. 14A-L show the homopolymer distributions found experimentally for each of the 12 bits of homopolymer addition.

FIG. 15 illustrates the cycle steps for template-independent DNA polymerase-mediated synthesis of homopolymer-encoded information polymers.

FIG. 16 shows a side view of a typical reaction zone in a 2D array of reaction zones, showing the hydrophobic patterning that isolates the liquid reaction droplets.

FIG. 17 shows a side view of a reaction zone with a controlled heating element or electrochemical element.

FIG. 18 shows the reaction zone showing the light-transmitting top cover.

FIG. 19 shows an exemplary device useful for addressable photoactivation of enzymatic DNA synthesis.

FIG. 20 shows the kinetics of UV light decaging of 3'-O-(2-nitro)-benzyl dATP.

21 shows optically controlled oligonucleotide extension. Enzyme reaction mixtures containing 3'-orthonitrobenzyl dATP were illuminated with a low power light source over various intervals such that the exposure time controlled the amount of decaging. The amount of available native dATP formed then controlled the average length of the polynucleotide tract.

FIG. 22 shows the structures of 3'-O-caging modifications that enable bichromatic light-mediated decaging.

FIG. 23 shows gel electrophoretic analysis of each of the 12 cycles of homopolymer synthesis using 3'-O-(2-nitro)-benzyl dATP and dCTP, dGTP, and dTTP.

FIG. 24 shows an electrophoretic analysis of an enzymatic synthesis reaction comparing incorporation-modulated dNTP analogs with unmodified dNTPs.

25 shows electrophoretic analysis of two successive cycles of incorporation rate-modulating dNTP analogs. The modified analog was used to form an N+1 homopolymer, followed by addition of an N+2 homopolymer. The homopolymer synthesis rate-modifying modifications were removed from the oligonucleotides prior to gel analysis.

26 shows an electropherogram of a TdT extension reaction showing homopolymeric rate controlling analog incorporation followed by a second cycle of homopolymeric rate controlling analog incorporation, where the modified nucleotide is followed by consecutive modified nucleotides.

FIG. 27 shows the modified dNTP analogs used in the information polymer composition.

FIG. 28 shows an electrophoretic analysis of information polymers composed of modified nucleotides.

FIG. 29 shows detection of an information polymer (P71) composed of modified nucleotides by translocation through a nanopore.

FIG. 30 shows detection of an information polymer (86B) composed of modified nucleotides by translocation through a nanopore.

DETAILED DESCRIPTION The present invention presents systems and methods for writing data to and reading data from nucleic acids having homopolymer tracts that correspond to units of digital data. By repeating each nucleotide in the data encoding sequence (e.g., 3-10 times), only the transitions between homopolymer tracts need to be observed in the sequencing read, which allows for lower fidelity, but higher throughput sequencing techniques that may result in cheaper implementation in nucleic acid data storage. The advantages of synthesizing nucleic acid homopolymer tract memory strands are: 1) the ability to make very long (5-10 kb) strands that allow the use of high throughput, long read DNA sequencing techniques for readout, 2) the ability to tolerate errors in the sequencing readout techniques, and 3) the ability to make nucleic acid memory strands at a much lower cost than that of traditional chemical synthesis methods. The use of homopolymeric nucleic acid memory strands is best realized in long (e.g., 5-10 kb) strands that can be efficiently produced using template-independent TdT enzymes or polymerase theta, where the homopolymer tract length can be controlled by varying the exposure time and ratio of dNTPs to polynucleotide memory strands.

Synthesis of homopolymers for encoding data by an enzymatically mediated approach is easily accomplished by using non-terminator natural or modified nucleotide triphosphates, allowing the simplest and most rapid method of DNA synthesis. One natural or modified nucleotide triphosphate is delivered to a reaction zone with nucleotidyl transferase, the reaction occurs, and then removed by washing with a buffer to complete one "write" cycle of data storage as illustrated in Figure 1. Data strand synthesis occurs in a completely aqueous environment without toxic or harmful chemicals, thus enabling a practical device suitable for large-scale data storage centers.

2 shows a method 101 of synthesizing a nucleic acid memory strand having homopolymer tracts according to certain embodiments. The method 101 includes a step 103 of creating an in-silico oligonucleotide sequence representing a data set. The data set may include digitized data that may represent text, images, video, audio, or any other piece of information that may be digitized. The oligonucleotide sequence may include any number of natural or modified nucleotides or their analogs, and may encode the data set using a base 2, base 3, base 4, or higher base scheme, depending on the number of unique nucleotides or analogs used in the memory strand. In a simple embodiment, the encoding scheme may correspond to a binary data scheme conventionally represented by a series of 0s and 1s, where one or more nucleotides or analogs may correspond to 0 and one or more other nucleotides may correspond to 1. A nucleic acid memory strand (e.g., RNA, single-stranded or double-stranded DNA) may then be synthesized 105 that includes a series of homopolymer tracts that each correspond in order to a nucleotide in the in silico oligonucleotide sequence. In certain embodiments, further steps include modifying one end of the memory strand (step 107), passing the memory strand through the nanopore (step 109), and modifying the other end of the strand (step 111) to prevent the end from passing through the nanopore.

Figure 3 shows a method 203 for reading data from a nucleic acid memory strand having homopolymer tracts. The steps of the method 203 include sequencing 203 a series of homopolymer tracts in the nucleic acid memory strand, converting 205 the sequence into a data set, converting the data set into a readable format (e.g., an image, video, audio clip, or piece of text), and displaying 209 the readable format data as desired (e.g., on a monitor or using a printer or other input/output device).

Preferably, the systems and methods of the invention use long strands of DNA (5-10 kb) that are either single-stranded or double-stranded and can occur naturally or can be produced by chemical or enzymatic synthesis. In certain embodiments, the nucleic acid memory strands can be enzymatically generated using TdT to create a series of homopolymer tracts that can be 2-10, 3-10, 4-10 nucleotides or longer. The homopolymer tracts can each consist of adenine (A), guanine (G), cytosine (C), or thymine (T). The sequence of alternating homopolymer tracts can be used to encode the data to be stored in the memory strand.

Each nucleotide homopolymer tract can represent various amounts of data depending on the number of bases used. The number of bits required to compose one byte (256 decimal) is defined by the following relationship: #bits/byte=8/(log2(n)), where n=the base used. Each tract can correspond to one bit if a 2-base encoding is used, or 1/4 of a byte if a 4-base encoding is used. In certain embodiments, DNA data strands can be composed of homopolymer tracts of 2-10 nucleotides (using a base 2 data set representation), allowing 333 bits to 100 bits to be represented in memory strands between 999 and 1000 bases long. In a preferred embodiment, the nucleobase encoding of the data can be such that a single homopolymer tract of one nucleotide is always adjacent to a homopolymer tract of a different nucleotide. For example, the encoding can be such that a homopolymer tract of adenine is never immediately preceded or immediately followed by another homopolymer tract of adenine. When two adjacent homopolymer tracts contain the same nucleotide, homopolymer tracts that are measurably longer than the average homopolymer tract representing a single nucleotide in the encoded data sequence can be synthesized. These longer tracts can be created through manipulation of the synthesis reaction described below, for example, by increasing the concentration of dNTPs in the reaction or increasing the reaction time. The exact length of the two adjacent identical homopolymer tracts need only be long enough to be uniquely distinguished from a single homopolymer tract using a readout device (i.e., a nanopore sequencer). In certain embodiments, a non-nucleotide homopolymer spacer can be added between the A, G, C, or T homopolymer tracts to clearly distinguish adjacent same nucleotide homopolymer tracts from each other. The use of A, G, C, and T homopolymer tracts allows for the creation of a 4-bit encoding space, which increases the density of data that can be stored in one continuous strand, rather than simply using two nucleotides (similar to 0 and 1 in a binary code). For example, four consecutive homopolymer tracts can encode 256 numbers (i.e., one byte) when A, G, C, and T are used in a base-4 scheme. In such an embodiment, 83 bytes or 25 bytes are represented in a nucleic acid memory strand 996 or 1000 nucleotides long when using homopolymer tracts 3 or 10 nucleotides long, respectively.

In various embodiments, base-8 or even base-12 coding schemes can be used through the incorporation of homopolymeric tracts of uniquely modified nucleotides or non-nucleotide analogs into the memory strand. These modified nucleotides or non-nucleotide analogs should generate unique digital signals on a readout device such as a nanopore sequencer or a single molecule ZMW sequencer. TdT, discussed below, can incorporate a wide range of modified dNTP analogs that can enhance the signal provided by a readout device such as a nanopore and thus can be used to generate nucleic acid memory strands with data encoded therein. Homopolymers of modified nucleotides (e.g., A ^* , G ^* , C ^* and T ^* or A ^** , G ^** , C ^** and T ^** ) can be synthesized using TdT and modified dNTP analogs of each of the four bases (e.g., dA ^* TP or dA ^** TP) to generate 8-bit or even 12-bit encoding schemes. Encoding higher bases (n) allows for data compression, resulting in a reduction in the number of DNA strands required to encode a given amount of information. The relationship that determines the number of DNA strands per GB of data as a function of homopolymer tract length, base (n), and synthesized strand length, as illustrated in Figure 4, is defined by: # strands/GB = (8/(log2(n)) ^{* 109 *} homopolymer length ^* 1/strand length.

The number of unique homopolymer tracts may only be limited by the ability of the readout technology (i.e., nanopore or ZMW single molecule sequencing) to determine one homopolymer tract from another. There are several reports in the literature of detection of homopolymers composed of unmodified nucleotides by detecting changes in ionic current during translocation through a nanopore (Venta et al, 2013; Feng et al, 2015). Modifications that alter the residence time of DNA in the nanopore generate distinguishable and characteristic ionic current signals. Singer et al 2010 and Morin et al 2016 use non-covalently linked bis-PNA or gamma-PNA functionalized with 5 kDa or 10 kDa PEG to enhance detection by nanopores. Liu et al 2015 selectively created adamantly 8-oxo G analogs to modify residence time and generate unique signals. Considering the tolerance of TdT to incorporate bulky modifications at N6 of dATP, N4 of dCTP, N2 or O6 of dGTP, and O4 or N3 of dTTP, acyl or alky modifications at these positions can be screened and selected to enhance the detection modalities of nanopore or ZMW single molecule sequencing technology. Detection can be improved through modified nucleotides that enhance differential current blockade in nanopores or enhance the residence time of modified nucleotides in the active site of DNA polymerase in ZMW single molecule approaches. Other natural and unnatural purine and pyrimidine nucleotide analogs can be used if they generate unique digital signals on readout devices such as nanopore or single molecule ZMW sequencers. Modifications at C5 or C7 of pyrimidines and purines, respectively, can be used if they generate unique digital signals on readout devices such as nanopore or single molecule ZMW sequencers. Suitable modified nucleotide triphosphates are selected to be rapidly incorporated during the enzymatic extension step and provide substitution-specific dwell times with the shortest possible homopolymer during the detection step. Examples of modifications to A, G, C and T bases suitable for expanding the bit encoding space include, but are not limited to, N6-benzoyl dA, N6-benzyl dA, N6-alkyl dA, N6-acyl dA, N6-substituted alkyl dA, N6-substituted acyl dA, N6-arylacyl dA, N6-substituted arylacyl dA, N2-alkyl-dG, N2-acyl dG, N2-arylacyl dG, N2-substituted alkyl dG, N2-substituted acyl dG, N 2-substituted aryl acyl dG, O6 alkyl dG, O4 alkyl dT, N3 alkyl dT, N3 acyl dT, O6 substituted alkyl dG, O4 substituted alkyl dT, C5-propargylamine dT, C5-propargylamine dC, C7-propargylamine dA, C7-propargylamine dG, substituted C5-propargylamine dT, substituted C5-propargylamine dC, substituted C7-propargylamine dA, substituted C7-propargylamine dG. Preferred embodiments of substitutions include covalent bonds that are completely stable to removal, except under the most extreme chemical conditions of pH, temperature and concentration of reactive species. Substitutions that can affect unique current blockages can include, but are not limited to, alkyl, heteroatom-substituted alkyl, aromatic hydrocarbons, alkyl-substituted aromatic hydrocarbons, heteroatom-substituted alkyl-substituted aromatic hydrocarbons, heteroatom-substituted aromatic hydrocarbons, benzyl, substituted benzyl, or combinations thereof. In some embodiments, the substitution can be polyethylene glycol composed of 2-450 monomer units. In some embodiments, substitutions composed of peptides or peptoids may be suitable for increasing homopolymer residence time in a specific and discriminable manner. The efficiency of incorporation of modified nucleotides by template-independent polymerases such as TdT can be modulated by the use of different metal ion cofactors, such as, but not limited to, Co++, Zn++, Mg++, Mn++, or a mixture of two or more different metal ions. Each modified nucleotide may require a different metal ion for optimal performance during enzymatic homopolymer synthesis.

Since long-term stability of DNA data strands is important, there is a distinct advantage to using non-purine-based homopolymers, since they are subject to depurination at low pH. In some embodiments, homopolymer bits may be composed of only a single nucleotide type (i.e., thymine) modified with two, three, four or more different chemical groups, resulting in homopolymer tracts that each produce a unique current block. Thus, one nucleotide labeled with four unique modifiers may replace the occurrence of A, G, C, T. Other embodiments are possible that use only one of the other three nucleotides with two, three, four or more different chemical groups.

Another embodiment uses a single nucleotide bit instead of a homopolymer bit, where the modified nucleotide analog is sufficient to produce a unique residence time for the passage of a single nucleotide through the nanopore. A single modified nucleotide bit is advantageous in that it allows for the greatest density of information per DNA strand, thus reducing the cost of DNA-based data storage.

The exact length of the homopolymer tract is not important, so long as the sequencing technology used for readout can clearly distinguish the start and end of one homopolymer tract from another. Although there are clear synthesis and storage density advantages in increasing the number of unique nucleotides or bases (including modified nucleotides or non-nucleotide analogs) used, and thus reducing the length required to capture a set amount of data, the lowest cost per DNA data storage synthesis can be achieved by using the four natural nucleotide dNTP monomers during enzymatic synthesis, since these reagents are widely used in the fields of molecular biology and sequencing and are produced in very large batches with the lowest manufacturing costs. The cost of producing dNTP analogs to increase the number of unique homopolymer tracts can be reduced as the use of DNA data storage increases and the scale of analog manufacturing is also increased.

Although any method of synthesizing homopolymer tract segments can be used with the systems and methods of the present invention, a preferred embodiment uses the template-independent enzyme TdT. TdT offers certain advantages in that it quickly and cheaply produces homopolymers with a Poisson distribution, where the average size of the homopolymers can be tightly controlled by the ratio of [dNTP] to the nascent oligonucleotide memory strand. In some embodiments, polymerase theta in the presence of Mn2 ⁺ can be used as a template-independent polymerase to synthesize homopolymer tract nucleic acid memory strands. In another embodiment, the length of the homopolymer tract segment can be controlled by delivering an excess amount of dNTP to the reaction zone and then removing the reactants after a carefully controlled time interval.

TdT has demonstrated the ability to synthesize homopolymer tracts of well-defined length by controlling the ratio of dNTP concentration to the concentration of the 3' end of the nucleic acid strand desired to be modified. Inkjet synthesis on an array-based format allows for very low-cost phosphoramidite synthesis, but the strands produced are limited to 100-200 bases in length, making them primarily suitable for relatively low-efficiency short-read sequencing readout techniques made at sub-femtomolar scales that must sacrifice some length to index the sequence and require post-synthetic amplification to provide sufficient material for subsequent readout.

A strand of single-stranded DNA synthesized according to the process of the present invention may benefit from the prevention of hairpins or dsDNA, either during synthesis or during readout. Hairpin formation may be prevented by modifying the exocyclic amine of one member of the A:T or G:C base pair to prevent hydrogen bonding necessary for base pairing. In some embodiments, the exocyclic amine may be modified by acylation or alkylation. Any simple and stable modification of the exocyclic amine of A, G or C that prevents base pairing may be used to prevent hairpin formation. In certain embodiments, the N6 of deoxyadenosine and the N2 of deoxyguanosine may be acetylated with an acetyl group that prevents base pairing. In some embodiments, the N6 of deoxyadenosine and the N4 of deoxycytidine may be modified to prevent base pairing and hairpin formation. In some embodiments, the O6 of deoxyguanosine or the O6 of deoxythymidine may be modified to prevent base pairing and hairpin formation. In some embodiments, the O4 of deoxythymidine or the N3 of deoxythymidine may be modified to prevent base pairing and hairpin formation. In some embodiments, modifications to A, G, C, or T to generate higher order base encoding schemes also serve the purpose of preventing base pairing and hairpin formation. In some embodiments, homopolymer bits may be composed of only a single nucleotide type (i.e., thymine) modified with two, three, four, or more different chemical groups, resulting in a unique current blockade and preventing the formation of intrastrand or interstrand duplex regions. In another embodiment, a thermostable version of TdT or another template-independent nucleotidyl transferase may be used to perform strand synthesis at elevated temperatures, thus preventing the formation of intrastrand or interstrand duplex regions.

Control of homopolymer tract length can be optimized for any of the dNTP analogs after determination and calibration of the incorporation rate of the analog to create a reproducible range of homopolymer tract lengths from 2 to 10 nucleotides long. The use of A ^* , G ^* , C ^* , and T ^* , as well as A ^** , G ^** , C ^** , and T ^** homopolymer tracts allows for the creation of 8-bit or 12-bit encoding, increasing the density of data that can be stored in one continuous strand rather than simply using two nucleotides to encode "0" and "1". Three consecutive homopolymer tracts can encode 256 numbers (i.e., one byte) when A, G, C, T, A ^* , G ^* , C ^* , and T ^* are used. In such an embodiment, there are 111 or 33 bytes in a 999 or 990 nucleotide long nucleic acid memory strand when using 3-nucleotide or 10-nucleotide long homopolymer tracts, respectively. Two consecutive homopolymer tracts can encode 256 numbers (i.e., one byte) if A, G, C, T, A ^* , G ^* , C ^* , T ^* , A ^** , G ^** , C ^** , and T ^** are used. In these embodiments, there are 166 or 50 bytes in a 996 or 1000 nucleotide long nucleic acid memory strand if 3 or 10 nucleotide long homopolymer tracts are used, respectively.

In certain embodiments, data can also be encoded into memory chain heteropolymer tracts of random sequence and defined composition to achieve higher levels of data compression. Heteropolymer stretches can be generated using enzymatic reactions using mixtures of different dNTPs, where dNTP stoichiometry is used to control the composition of the heteropolymer tracts. The number and type of heteropolymer tracts is limited only by the combination of dNTP analogs and the ability of the detection modality to distinguish the composition of the different tracts. For m dNTP analogs, there are (m ² -m)/2 binary combinations for heteropolymer formation. A detection modality that can distinguish two different levels of tract composition for each binary combination (e.g., tracts where analogs A and B are present in approximately a 2:1 ratio, respectively, and tracts where they are present in a 1:2 ratio) would allow data to be encoded at base m ² rates from a set of m analogs, effectively doubling the coding capacity of the memory chain. FIG. 5 illustrates the data that can be stored in a memory chain as a function of the number of available dNTP analogs using either homopolymer and binary heteropolymer-based encoding schemes with two levels of tract composition.

The data encoding strands of the present invention may not necessarily require precisely defined homopolymer lengths, since they only need to be long enough (approximately 2-10 nucleotides) to allow unambiguous discrimination of the transition between homopolymer tract segments by high-throughput DNA sequencing techniques. Existing next-generation sequencing-by-synthesis (SBS) systems can easily determine the transition between two adjacent homopolymer tracts. Again, the exact length of the homopolymer tract is not critical for accurate detection of homopolymer bits. The use of tracts of the same nucleotides offers an advantage in overcoming the most common errors in current SBS platforms: insertions and deletions. A deletion of one nucleotide in a homopolymer tract longer than 2 nt is still interpreted as a true homopolymer. Similarly, an insertion of a single nucleotide in a homopolymer tract will not be misinterpreted as two adjacent homopolymers, since an insertion of more than one nucleotide between SBS is an unlikely event. This sequencing error tolerance offers the advantage of reducing the sequencing depth required to ensure correct decoding of the information stored by the DNA data strand. Existing nanopore systems can easily distinguish homopolymeric tracts of A, G, C, or T from one another based on their differential current blockade. In certain embodiments, single-molecule ZMW sequencing can be used to determine the linear order of homopolymeric tracts on a linear strand. The use of either sequencing technique may require the DNA initiator to have properties compatible with the sequencing readout technique, such as a self-complementary hairpin at the 5' end of the synthesized single-stranded memory strand to provide a primer for single-molecule ZMW sequencing. Nanopore sequencing techniques may also require a self-complementary hairpin at the 5' end of the strand to provide a "start" data mark. In various embodiments, the readout technique can be any next-generation sequencing method, such as those provided by Illumina (San Diego, Calif.). In some embodiments, the readout or sequencing technique can be mass spectrometry-based. The technique-specific error rate of the readout technique is not important as long as the technique can unambiguously detect the transition between two different homopolymer tracts and/or, when two identical homopolymer tracts are adjacent to each other, the difference between one homopolymer tract length and one 2× length.

Certain readout techniques may be preferred over others based on the specific application of the invention. Techniques such as nanopore sequencing may be non-destructive, may leave the nucleic acid memory strand intact, and may be suitable for multiple readout cycles. Readout techniques that rely on sequencing by synthesis (SBS), such as ZMW single molecule, may require a post-readout operation (i.e., strand separation by melting) to generate a copy of the original template strand and remove the complementary strand, returning the original nucleic acid memory strand to its initial state ready for subsequent cycles of readout. Other readout techniques, such as mass spectrometry, are destructive and deplete the pool of nucleic acid memory strands after repeated cycles of sampling and readout.

In various embodiments, the nucleic acid memory strand may include a "stopper." A "stopper" may be a polymeric construct that prevents the passage of single-stranded or double-stranded nucleic acid through the nanopore (Manrao, et al., 2012, Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase, Nature Biotechnology 30, 349-353, incorporated herein by reference). Proteins such as phi29 DNA polymerase are large enough not to be pulled through the larger pore (about 6.3 nm) on the cis side of the protein nanopore. The pore diameter on the small side is estimated to be about 1.2 nm wide. Stoppers may be used on either the 5' or 3' end of the nucleic acid memory strand of the invention. In some applications, it may be desirable to have stoppers on both the 3' or 5' end of the nucleic acid molecule. The stopper may consist of a hairpin (stem-loop) structure with either a protruding 5'-overhang or a 3'-overhang to which the information-encoding nucleic acid is covalently attached. If the stopper consists of a hairpin, the length of the ds stem may be long enough to resist any melting force exerted thereon by the electric field used to translocate the memory strand through the nanopore. In certain embodiments, one base of the double-stranded stem region may be cross-linked to its cognate base that forms a base pair such that the double-stranded stem portion of the hairpin is unable to melt under the influence of the force exerted thereon by the electric field that translocates the remainder of the molecule through the nanopore. To utilize a hairpin stopper for TdT-mediated nucleic acid memory synthesis according to certain embodiments, the stopper may have a 3'-overhang of sufficient length (i.e., >10 nucleotides) to allow binding of TdT for template-independent synthesis.

The stopper may consist of a non-nucleotide polymeric construct that can be added to either the 3' or 5' end of the nucleic acid molecule. The construct may be synthesized by direct conjugation of a polymeric species onto the 3' end of the nucleic acid by a polymerase or a transferase such as TdT (Sorensen, et al., 2013, Enzymatic Ligation of Large Biomolecules to DNA, ACS Nano, 7(9):8098-8104, incorporated herein by reference) or by incorporation of a functionalized nucleotide that allows specific modification of the nucleic acid via its functionality (Winz, et al., 2015, Nucleotidyl transferase assisted DNA labeling with different click chemistries, Nucleic Acids Res. 43(17):e110, incorporated herein by reference). The 5' end stopper can be easily introduced at the time of chemical synthesis of the oligonucleotide adaptor, either through direct synthesis of the hairpin or through secondary modification of a functional handle introduced as the last step of the 3' to 5' oligonucleotide synthesis, and used as an initiator. Alternatively, the 5' end stopper can be constructed by attaching an oligonucleotide initiator via its 5' end to a magnetic or non-magnetic bead or particle or nanoparticle, enzymatically synthesizing a memory strand containing a homopolymer tract, and then leaving the memory strand attached to the magnetic or non-magnetic bead or particle or nanoparticle.

The stopper can be further modified to allow cleavage of the stopper from the remainder of the molecule to allow the nucleic acid strand to passively diffuse out of the nanopore, or to allow the nucleic acid strand to be translocated out of the nanopore via application of a voltage, thus allowing the strand to be retrieved.

In certain embodiments, template-independent polymerases or transferases can be used to modify pre-synthesized strands of nucleic acid to enable the use of nanopore devices as "write once read many" type memory devices. Some of the inherent problems associated with the use of nanopore devices as DNA sequencers are the high error rates they produce due to the poor discrimination of the nanopores. This can be attributed to the speed of translocation through the pore or the fact that the approximate depth of the nanopore is 8 nm, allowing multiple bases to be present in the pore simultaneously. The homopolymer memory strands of the present invention address this issue through the use of homopolymer repeats, reducing the need for strict sequencing accuracy. In certain embodiments, the shortcomings of nanopore sequencing can be addressed by implementing a hairpin adapter at one end of the double-stranded DNA memory strand, so that during the translocation and base calling process, each sense of the DNA memory strand can be read such that reading each individual base and its complementary strand can offset the error rate of reading each base only once. In certain embodiments, the fidelity of nanopore sequencing can be increased by appropriate modification of each end (5' and 3') of a single-stranded or double-stranded nucleic acid molecule with bulky appendages (e.g., proteins or solid states) that do not translocate across the pore. The molecule can then be trapped in the pore and translocated forward and backward multiple times to allow multiple reads of the same molecule in the same pore, thus reducing the sequencing error rate by the square of the number of reads (if the sequencing read errors are due to stochastic origins).

Transferases such as TdT can be used to append large, bulky modified nucleotide analogs to the 3' end of a DNA molecule. In certain embodiments, a WORM nanopore memory device can be generated using the following steps: (1) generate a single molecule of DNA that encodes specific information in any of the high-density encoding schemes discussed above, and covalently modify the 5' end with a bulky molecular construct that prevents complete translocation of the DNA molecule through the nanopore; (2) thread the DNA molecule through the nanopore until the 5'-modified end contacts the nanopore and cannot be translocated any further; (3) use TdT and the modified nucleotides to effectively trap the molecule within the torus of the nanopore at the 3' end of the DNA molecule. (4) covalently attaching one (or more) bulky nucleotide analogs ("stoppers") to the 3' end; (5) reversing the polarity of the current to the nanopore to remove any DNA molecules that are not 3'-modified, thus creating a pure population of "trapped" (5'- and 3'-modified) nucleic acid strands; (6) removing any untrapped nucleic acid from the vicinity of the nanopore via washing or other means; (7) reading the "trapped" DNA strands in either or both directions using an applied voltage (potentially reading multiple times to reduce the error rate to an acceptable level). In various embodiments, step 6 can consist of a voltage-induced "read" in one direction and rapid translocation in the opposite direction to "unwind" the data-encoding nucleic acid through the nanopore, followed by another voltage-induced "read" in the original direction. This cycle of "read"-"unwind"-"read" can be repeated as many times as desired.

In some embodiments, the trapped nucleic acid strand can be read during translocation in either direction. In some embodiments, the trapped strand can be translocated to one end of the molecule (either 5' or 3') and read in the opposite direction, so the polarity of the read can provide a more accurate read.

In certain embodiments, circularized nucleic acid memory strands can be produced using the synthesis method described above followed by circularization. The circularized strands can include bulky macromolecules or specific homopolymer sequences, where the ends of the synthesized strands are spliced to specify start and stop points for data reading. Start and stop homopolymer sequences can also be used in linear nucleic acid strands. The circularized strand 305 can be threaded between two adjacent nanopores (306 and 309) such that the circularized strand 305 is physically trapped between two nanopores (306 and 309) located on a single membrane 303 as shown in FIG. 6. The circularized memory strands can encode digital information as either a sequence of a single nucleotide, a homopolymer tract sequence, a sequence of modified nucleotide analogs, or some combination thereof. One nanopore 309 may be used to generate an electrical signal when the information-encoding memory strand is translocated through the pore, while the other nanopore 307 may simply function as a portal to allow the DNA molecule to return to the cis side of the membrane 303 and the first nanopore 309. One advantage of this scheme is that the information-encoding strand may be recycled for repeated reading and may be read multiple times, thus reducing any possible read errors.

Other embodiments may encode data in the memory strand such that the data can only be accessed under a specific set of conditions. In such cases, the memory strand is at least partially composed of nucleotides containing modifications attached to cleavable linkers. The modifications (e.g., chemical protecting groups) and linkers may be selected such that the current blockade is different from the sequence encoding the data when the polymer tract translocates through the nanopore without the correct treatment. Figure 7 outlines a scheme using disulfide and amide linked modifications to dG nucleotides and illustrates how the current blockade and the data encoded in the memory strand may change in response to treatment conditions. G ^* and G ^** are structurally similar in size and flexibility and may produce similar current blockade on the nanopore platform, but are removed under different conditions. Although ^G* and G ^*** are structurally different, these modifications share the same removal conditions. Other embodiments may use other modifications or linkers that are cleavable using different treatments such as specific wavelengths of light, acidic or alkaline pH, oxidative or reductive conditions, or sequence-specific nucleases. Some embodiments may use the presence or absence of memory chain modifications for encryption or as a chemical marker of previous access or modification of data. Most linker cleavage reactions are effectively irreversible, and therefore this approach is best suited for write-one read many systems, where a single molecule may be sufficient to encode data without redundancy.

Many possible information encoding schemes useful in the readout schemes of the present invention are possible and would be apparent to one of skill in the art based on this disclosure.

Synthesis can be accomplished using acoustic delivery of droplets into the wells of a plate (e.g., a 1536-well plate of 1.5 μL each). In various embodiments, the nucleic acid memory strands can be synthesized on beads or magnetic beads or surfaces and, depending on the application, can be left on the beads or magnetic beads or surfaces or removed from the synthesis support after full-length synthesis is complete.

In certain embodiments, a system for the synthesis of long (5-10 kb) data strands may use inkjet delivery to an array of wells (e.g., multiple nanoliter-volume wells). In other embodiments, multiple pneumatically controlled actuators may be positioned above each well to simultaneously deliver reagents to each location of the array. Each actuator is served by a selector valve that selects between each of two or more nucleotides or modified nucleotides formulated with a template-independent polymerase that are used to specify a bit of the DNA data strand. One or more additional selector valve ports are dedicated to one or more wash reagents, as needed. The array of nanoliter-volume wells may be open at both ends, so long as the diameter of the wells is such that the delivered liquid is trapped by capillary action within the length of the open-ended wells. After each round of nucleotide-enzyme formulation is delivered to the open-ended wells, each well is rinsed with the reaction mixture, thus preparing the array for the next cycle of enzymatic synthesis, and a rinse reagent or enzyme reaction stop reagent may be flowed across and through the lower opening of the array of wells. In other embodiments, a vacuum source is used to quickly remove one reagent from the capillary nanowell prior to delivery of the next reagent.

Certain embodiments may use highly parallel nanofluidic chambers with valve-controlled reagent delivery. An exemplary microfluidic nucleic acid memory strand synthesis device is shown in FIG. 8 for illustrative purposes, not to scale. A microfluidic channel 255 including a regulator 257 couples a reservoir 253 to a reaction chamber 251, and an outlet channel 259 including a regulator 257 removes waste from the reaction chamber 251. A microfluidic device for nucleic acid memory strand synthesis may include, for example, a channel 255, a reservoir 253 and/or a regulator 257. Nucleic acid memory strand synthesis may occur in a microfluidic reaction chamber 251 that may include several anchored synthesized nucleotide initiators, which may include beads or other substrates capable of releasably binding polynucleotide initiators, anchored or bound to the inner surface of the reaction chamber. The reaction chamber 251 may include at least one intake channel and one outlet channel 259 so that reagents can be added to and removed from the reaction chamber 254. The reaction chamber 251 must be temperature controlled to maintain optimal and reproducible enzymatic synthesis conditions. The microfluidic device may contain a reservoir 253 for each respective dNTP or analog used in the memory strand coding scheme. Each of these reservoirs 253 may also contain an appropriate amount of TdT or any other enzyme that extends DNA or RNA strands in a template-independent manner. Additional reservoirs 253 may contain reagents for washing or other tasks.

The reservoirs 253 may be coupled to the reaction chambers 254 via separate channels 255, and the flow of reagents into the reaction chambers 254 via each channel 255 may be individually regulated through the use of gates, valves, pressure regulators or other means. Flow from the reaction chambers 254 via outlet channels 259 may be similarly regulated. The reservoirs 253 may hold dNTPs, modified dNTPs or any analogs thereof suspended in a fluid at known concentrations, such that the concentration of the reagents may be precisely controlled based on the volume of reagent flowed into the reaction chambers 254. Thus, the length of each homopolymer tract may be managed through control of the reagent concentrations.

In certain instances, the reagents, particularly the dNTP and enzyme reagents, may be recycled. The reagents may be drawn back from the reaction chamber 254 into their respective reservoirs 253 through the same channel 255 they entered by inducing backflow using gates, valves, vacuum pumps, pressure regulators or other regulators 257. Alternatively, the reagents may be returned from the reaction chamber 254 to their respective reservoirs 253 through a separate return channel. The microfluidic device may include a controller capable of operating the gates, valves, pressure or other regulators 257 described above.

An exemplary microfluidic nucleic acid memory strand synthesis reaction may include flowing the desired dNTP (used throughout to refer to any component molecule used to encode data in the nucleic acid memory strand of the present invention) reagent into the reaction chamber 254 at a predetermined concentration (calculated to produce a desired homopolymer length) for a predetermined amount of time before removing the NTP reagent from the reaction chamber 254 via the outlet channel 259 or return channel (not shown); flowing a wash reagent into the reaction chamber 254; removing the wash reagent from the reaction chamber 254 via the outlet channel 259; flowing the next NTP reagent into the desired memory strand sequence under conditions calculated to achieve the desired homopolymer tract ratio; and repeating until the desired nucleic acid memory strand is synthesized. After the desired nucleic acid memory strand is synthesized, it may be released from the reaction chamber anchor or substrate and collected via the outlet channel 259 or other means.

Due to the significant number of homopolymer encoded DNA strands required to encode a usable amount of data, a highly parallel method of DNA synthesis is required. In some embodiments, as shown in FIG. 9, a flow cell (9-1) containing an array of wells is formed on a suitable substrate by patterning horizontal and vertical stripes of hydrophobic material (9-2) to form a plurality of hydrophilic wells (?) bounded by hydrophobic regions. Typical dimensions of the hydrophilic wells can be 300×300 nm to 1000×1000 nm. This hydrophilic array forms the floor of the flow cell, with a gap of suitable dimensions between the floor and an optically transparent cover. A solution of cold (i.e., below the optimal enzyme-specific temperature) nucleotidyl transferase, one of the native or modified nucleotide triphosphates, and any required cofactors is flowed into the flow cell via an inlet (9-3) such that upon cessation of fluid flow, the enzyme-nucleotide triphosphate solution bead up into spatially defined droplets (9-4) positioned above each hydrophilic region. The IR source (9-5) projects a beam (9-7) through a shaped lens (9-6) onto a DLP (digital light projection) device (9-8), which is used to simultaneously direct the IR beam (9-9) to each of the hydrophobically constrained droplets (9-4) to which a specific nucleotide is selected to be added, resulting in rapid heating of the polymerase extension reaction mix to a temperature for maximum enzyme activity over a defined period of time to synthesize a homopolymer of the desired length. After some well-defined reaction time, the IR source is switched off and a cold rinse buffer is rapidly injected into the flow cell and drained through the outlet (9-10), thus quenching the reaction and terminating one "write" cycle. This sequence of steps is repeated multiple times for each "write" cycle, such that each nascent data strand is randomly accessed according to its spatial location and selected nucleotides are added until a full-length homopolymer data strand is completed. In some embodiments, a DLP device with a 1920x1080 directional mirror can be used to simultaneously randomly access approximately 2M synthesis locations in a synthesis flow cell. In some embodiments, the template-independent polymerase used is thermophilic with an optimal reaction temperature well above room temperature, so that enzymatic activity is minimized in the hydrophobically constrained droplets prior to rapid heating of the droplets by the IR source. In some embodiments, the bottom surface of the flow cell bearing the hydrophobically defined hydrophilic wells abuts a cooling device that maintains the droplets in the hydrophobic wells at a reduced temperature to prevent enzymatic activity until the temperature is increased by the IR source.

In another embodiment, the flow cell is composed of an array of wells formed on a suitable substrate by patterning horizontal and vertical stripes of hydrophobic material forming a plurality of hydrophilic spots bounded by hydrophobic regions. Typical dimensions of the hydrophilic spots can be 300×300 nm to 1000×1000 nm. Each hydrophilic spot is located on an individually addressable CMOS heater. This hydrophilic-CMOS heater array, with a gap of suitable dimensions between the floor and the optically transparent cover, forms the floor of the flow cell. A cold (i.e., lower than the optimal enzyme-specific temperature) solution of nucleotidyl transferase and one of the natural or modified nucleotide triphosphates is flowed through the flow cell such that upon cessation of fluid flow, the polymerase extension reaction solution beaded into spatially defined droplets positioned above each hydrophilic region with an associated CMOS heater. Each of the hydrophobically constrained droplets to which a particular nucleotide is selected to be added is rapidly heated to a temperature for maximum enzyme activity for a defined period of time to synthesize a homopolymer of the desired length. After some well-defined reaction time, the heater is switched off and a cold rinse buffer is rapidly injected into the flow cell, thus quenching the reaction and terminating one "write" cycle. This sequence of steps is repeated multiple times for each "write" cycle, such that each nascent data strand is randomly accessed according to its spatial location and selected nucleotides are added until a full-length homopolymer data strand is completed. In some embodiments, the enzymes used are thermophilic with an optimum temperature well above room temperature, so that unwanted nucleotide addition in hydrophobically constrained droplets is unlikely prior to rapid heating of the droplets by the CMOS heater.

As those skilled in the art will recognize, as necessary or best suited for the systems and methods of the present invention, the systems and methods of the present invention may include computing devices as shown in FIG. 10, which may include one or more processors 309 (e.g., central processing unit (CPU), graphics processing unit (GPU), etc.), computer-readable storage devices 307 (e.g., main memory, static memory, etc.), or combinations thereof, in communication with each other via a bus. The computing devices may include mobile devices 101 (e.g., mobile phones), personal computers 901, and server computers 511. In various embodiments, the computing devices may be configured to communicate with each other via a network 517.

The computing device may be used to control synthesis of memory strands, reading of sequenced memory strands, and compilation or conversion of data between human or machine readable formats, digitized data, and nucleic acid sequences during other steps described herein. The computing device may be used to display data in a readable format.

Processor 309 may include any suitable processor known in the art, such as a processor sold under the trademark XEON E7 by Intel (Santa Clara, Calif.) or a processor sold under the trademark OPTERON 6200 by AMD (Sunnyvale, Calif.).

The memory 307 preferably includes at least one tangible, non-transitory medium capable of storing: one or more sets of executable instructions (e.g., software embodying any methodology or functionality found herein) for causing the system to perform the functions described herein; data (e.g., data encoded in a memory chain); or both. Although the computer-readable storage device may be a single medium in an exemplary embodiment, the term "computer-readable storage device" should be interpreted to include a single medium or multiple media (e.g., centralized or distributed databases and/or associated caches and servers) that store instructions or data. The term "computer-readable storage device" should be interpreted accordingly to include, without limitation, solid-state memory (e.g., subscriber identity module (SIM) cards, secure digital cards (SD cards), microSD cards or solid-state drives (SSD)), optical and magnetic media, hard drives, disk drives, and any other tangible storage medium.

Any suitable service, such as Amazon Web Services, memory 307 of server 511, cloud storage, another server, or other computer-readable storage may be used for storage 527. Cloud storage may refer to a data storage scheme where data is stored in a logical pool and the physical storage may span multiple servers and multiple locations. Storage 527 may be owned and managed by a hosting company. Preferably, storage 527 is used to store records 399 as needed to perform and support the operations described herein.

Input/output devices 305 according to the present invention may include one or more of a video display unit (e.g., a liquid crystal display (LCD) or cathode ray tube (CRT) monitor), an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse or trackpad), a disk drive unit, a signal generating device (e.g., a speaker), a touch screen, buttons, an accelerometer, a microphone, a cellular radio frequency antenna, a network interface device which may be, for example, a network interface card (NIC), a Wi-Fi card or a cellular modem, or any combination thereof.

Those skilled in the art will recognize that any suitable development environment or programming language may be used to enable the operability described herein for the various systems and methods of the present invention. For example, the systems and methods herein may be implemented using Perl, Python, C++, C#, Java, JavaScript, Visual Basic, Ruby on Rails, Groovy and Grails, or any other suitable tools. For computing device 101, it may be preferable to use native xCode or Android Java.

FIG. 11 shows polyacrylamide gel electrophoresis analysis of two different single homopolymer tracts made via enzymatic synthesis. Lane A is a sample of the 20-mer starting oligonucleotide used in all following lanes. Lane B is a sample from a TdT reaction containing a 20-mer oligonucleotide and the irreversible terminator ddATP showing the formation of a 21-mer. Lane C is a sample from a TdT reaction containing a 20-mer and the natural nucleotide dATP after 1 min at 37° C. Lane D is a sample of the same reaction mixture in lane D after 5 min at 37° C. Lane E is a sample of the same reaction mixture in lane C after 15 min at 37° C. These three lanes show homopolymer length control due to consumption of input dATP at approximately 5 min during the TdT extension reaction, and no homopolymer growth is observed between 5 and 15 min. Lane F is a sample from a TdT reaction containing a 20-mer oligonucleotide and the nucleotide analog N6-benzoyl-dATP after 1 min at 37° C. Lane G is a sample of the reaction mixture in lane F after 5 min at 37° C. Lane H is a sample of the same reaction mixture in lane F after 15 min at 37° C. Although there are qualitative differences between the lengths of the ^dABz homopolymers formed in lanes F-H, the same length control is also demonstrated when using the N6-modified dATP analog.

Writing digital data into molecular storage formats based on molecular approaches offers advantages over currently used storage media such as tapes or disks. DNA-based storage has sparked interest due to the high information density achievable, extremely long lifetimes, and low energy consumption during dormant periods. To date, most attempts to use synthetic DNA as a storage medium have involved chemical synthesis using the common phosphoramidite method.

DNA-based data storage may require a much larger number of strands than are currently synthesized for the existing research market. Any synthesis technique (chemical or enzymatic) that relies on nucleotide blockage or removal of terminators imposes additional steps and complexity because reagents must be delivered to the array of synthesis features (i.e., wells or spots) in an addressable manner to direct the correct nucleotide to the correct location on the array. Array-based methods of synthesis can be performed in one of several ways: 1) bulk delivery of activated reactants followed by selective removal of blocking groups, or 2) addressable delivery of activated reactants followed by bulk blocking group removal, or 3) bulk delivery of inactive reactants followed by addressable activation. Addressable delivery of reagents to large (10 ⁴ -10 ⁶ ) 2-D arrays is commonly achieved using inkjet deposition to each desired location. If the data encoding scheme uses four nucleotides, four separate writing heads must be used and indexed in a complex X-Y mechanical manner. Furthermore, the use of inkjet delivery limits the dimensions between features (wells or spots) to low tens of microns. A challenge in this process, however accomplished, is to reduce the step time for each synthesis cycle.

In certain embodiments, the systems and methods of the invention may include the steps of delivering an inactive reaction mixture to all features on a 2-D array, then selectively activating only those features that require the addition of A or G or C or T. Addressable methods of delivery, activation, or blocking group removal that do not rely on mechanical motion are preferred. A preferred embodiment uses bulk delivery of reactants and selective activation of specific synthetic features by removal of blocking groups from nucleotide analogs, which then allows for rapid DNA polymerase-mediated incorporation and formation of homopolymeric bits as shown in FIG. 15, thus enabling highly parallel and rapid synthesis of homopolymer-encoded nucleic acid memory strands. Several other advantages exist for homopolymer-encoded nucleic acid information polymers: 1) homopolymer-encoded bits overcome the error profile associated with next-generation sequencing, 2) the resulting polymers are non-natural nucleic acids and therefore cannot be diverted for bioterrorist activities.

Delivery and selective activation of template-independent polymerase DNA synthesis can be performed sequentially (deliver A -> addressably activate and initiate homopolymer synthesis -> wash; deliver C -> addressably activate and initiate homopolymer synthesis -> wash; deliver G -> addressably activate and initiate homopolymer synthesis -> wash; deliver T -> addressably activate and initiate homopolymer synthesis -> wash) or in parallel (deliver all four nucleotides to all features simultaneously -> addressably activate A, C, G, T simultaneously or sequentially -> wash). In this manner, the synthesis cycle becomes highly efficient and involves only three steps: reactant delivery, incorporation reaction activation, then wash before the next cycle begins. The reaction is stopped by rapid removal of the reactants with a gas or liquid, or by rapid delivery of a quenching reagent. In a preferred embodiment, the quenching reagent is a metal chelator.

A preferred design for a device that can be used to synthesize homopolymeric bit-encoding memory strands consists of a flow cell composed of a 2-D array of hydrophobically patterned wells appropriately modified to support template-independent enzymatic synthesis. After delivering reactants to the 2-D array of hydrophobic wells, the liquid beads up to define spatially distinct reaction zones, as shown in Figure 16.

A preferred embodiment is one in which the bottom surface of the well, surrounded on all four sides by hydrophobic patterning, is modified with a covalently attached oligonucleotide initiator in a 5'->3' orientation, with the 5' end attached to the bottom surface of the well. In another embodiment, the well is physically formed by etching a depression into the bottom surface of the flow cell, in which case the covalently attached oligonucleotide initiator is covalently attached to the surfaces (bottom and sides) of the well. In some cases, the well is open at the bottom to allow for liquid flow through the well. In other cases, the well is closed at the bottom.

Nucleotide analogs protected at the 3'-OH are generally inactive with commercially available or WT TdT enzymes (U.S. Pat. No. 10,059,929). Thus, a mixture of 3'-blocked dNTP analogs, TdT protein and appropriate cofactors can be mixed together in the presence of initiator oligonucleotides at 37°C with little to no homopolymer formation. Once the 3'-OH is "decased" (i.e., rendered unblocked) by removal of the protecting or blocking group, the resulting nucleotide is available for free running incorporation and homopolymer formation. Decasing that can be achieved by "delivery-free" methods is preferred. Analogs constructed to allow addressable 3'-OH decasing by electrochemical generation of activation energy, e.g., light, heat, pH change, or administration of or exposure to reducing agents are preferred. Each of these de-caging or unblocking reactions can be accomplished in an appropriately constructed flow cell using either mechanically directable light through a light-transmitting cover (e.g., FIG. 17), individually addressable heaters (e.g., FIG. 18), electrochemically induced pH changes, or the creation of reducing conditions.

Each of the illustrated flow cell designs is compatible with the method of addressable decaging of nucleotides contained in droplets constrained by hydrophobic patterning around them. Figure 19 illustrates an apparatus that can be used with a flow cell designed for the use of photodecaged nucleotide analogs. Other embodiments and configurations known to those of skill in the art are possible for this purpose.

Each decaging mechanism requires a nucleotide analogue specifically designed for the physicochemical process selected for use in the system:

In some embodiments, the nucleotide analogue suitable for light-mediated decaging is 3'-O-(2-nitro-benzyl)-dNTP. In some embodiments, the nucleotide analogue suitable for heat-mediated decaging is 3'-O-(tetrahydrofuranyl)-dNTP. In some embodiments, the nucleotide analogue suitable for reduction-mediated decaging is 3'-O-methyl-dithiomethyl-dNTP. Many other 3'-OH protecting groups are suitable, so long as the resulting 3'-OH modified nucleotide analogue is not a substrate for template-independent polymerases and is easily removed by "delivery-free" methods, e.g., light, heat or electrochemically generated reactants. Insofar as the 3'-O modifications described herein are explicitly stated to be substrates for template-independent polymerases and are referred to as reversible terminators, the composition of these dNTP analogues differs from those described in WO2016/034807. The subject of the present invention is not explicitly a substrate for polymerase, but rather a 3'-O-blocking group that functions to cage dNTPs until it is removed to allow polymerization to proceed. Mathews AS et al (2016) describe the use of 3'-O-(2-nitrobenzyl)-dNTP analogs as reversible terminators for the controlled enzymatic synthesis of natural non-homopolymeric oligonucleotides. They explicitly teach the use of such nucleotides as substrates for DNA synthesis by using extremely long (approximately 1 hour) enzymatic reaction times, and furthermore, as reversible terminators, in contrast to the subject of this patent, which teaches the use of these analogs as caging groups to initiate enzymatic polymerization of multiple nucleotides. Initiation of free-running homopolymer synthesis can be achieved by methods other than caged dNTP analogs. Some embodiments may use fluid pulses of polymerase to initiate and terminate ssDNA synthesis, as described in Church 9,928,869 (2018). In Reza et al WO2017/196783, template-dependent enzymatic synthesis is initiated by activation of the polymerase by electrochemically generated pH changes. Modified nucleotide analogs containing 3'-O-reversible terminators are described, but neither patent teaches activation of homopolymer synthesis using decaging of caged dNTP analogs. Church 9,928,869 (2018) describes free-running homopolymer synthesis using natural dNTPs and controlling the length of homopolymer formed by reaction duration. Lee HR et al (2018) describe the use of mechanical delivery to deposit natural nucleotides into multiple reaction zones on a 2D array and control the length of homopolymer formation by actively destroying unreacted unmodified dNTPs with apyrase. In some embodiments of the subject matter of this patent, homopolymeric rate-modifying modified dNTP analogs with an unmodified 3'-OH are used in combination with either fluid pulse or mechanical XY delivery or pH-activated template-independent DNA polymerase.

Figures 20 and 21 show the kinetics of light-mediated decaging. Once converted to a substrate for the polymerase, the 3'-caged dNTP in the template-independent polymerase reaction mixture is polymerized into a nascent homopolymer strand. Complete conversion of the caged dNTP to an uncaged (substrate) dNTP is not required, since available uncaged dNTPs are readily incorporated by the polymerase. Once decaged, the desired homopolymer length is controlled by controlling the concentration of the decaged dNTP produced and/or the time of the enzymatically mediated incorporation reaction.

Figure 22 shows an example of two nucleotide analogs caged with 3'-O modifications that can be decaged with visible wavelengths of light and exhibit tunable photophysical properties (Peterson J.A., et al J. Amer. Chem. Soc. 2018 140:7343-6), allowing for the simultaneous introduction, decaging, and synthesis of two different homopolymeric chain sequences. In some embodiments, nucleotide analogs modified with two different 3'-caging species that can be decaged with two different wavelengths of light are delivered simultaneously to a 2D array of synthesis locations. In another embodiment, four dNTP analogs modified with four different photoremovable 3'-caging species are delivered simultaneously to a 2D array of synthesis locations and decaged with four different wavelengths of light. The decaging reaction can be performed sequentially for a set of locations on the 2D array, followed by delivery and subsequent sequential decaging of the two remaining dNTPs, thus completing one round of increasing the length of all information polymers by one nucleotide. In an alternative embodiment, four dNTP analogs modified with four different 3'-caging species can be delivered simultaneously and decaged by simultaneous exposure of each synthetic trait to one of four different wavelengths of light. A hardware configuration such as that shown in FIG. 19 can be used for multi-color decaging by incorporation of two or more wavelength-specific light sources and an appropriate shutter mechanism to direct one of the two or more light sources onto a 2D light directing system.

Figure 23 shows the synthesis of a 12-bit information strand consisting of successive cycles of homopolymer synthesis using dCTP, dGTP, dTTP interspersed with UV light decased 3'-oNBn-dATP in cycles #1, 6, 8, 10, 12. The incorporation reaction can be terminated by several methods, including but not limited to, rapid removal of the enzyme reaction components by introduction of a bolus of either gas or liquid. In some embodiments, the liquid simply rinses away the reaction components, while in other embodiments, the rinse liquid contains an active quenching agent such as EDTA or other enzyme inhibitors.

In another embodiment, the method of caging a dNTP can be mediated by a steric mechanism involving a modification to the nucleotide base instead of the 3'-OH. In a preferred embodiment, a nucleotide with an unmodified 3'-OH is modified at N6, N4, N2, O4 of A, C, G or T, respectively, with a removable steric blocking group (incorporation blocker) that cages the dNTP. Homopolymer synthesis can be initiated by either light-, heat- or reduction-mediated cleavage of the steric blocking group, thus "uncaging" the natural nucleotide suitable for free-running homopolymer synthesis.

In some embodiments, the modified nucleotide is composed of a linker that contains one moiety that renders the dNTP caged, and therefore incapable of incorporation, until removed, and another moiety that remains covalently attached throughout the synthesis of multiple homopolymer tracts. In some embodiments, the purine or pyrimidine base is modified at two positions, one modification that cages the nucleotide and prevents enzymatic incorporation, while the other modification remains covalently attached throughout the synthesis of the polymer; each modification is removable by a different mechanism that allows for selective removal of each. In some embodiments, the purine or pyrimidine base is modified at two positions, each modification is removable by a different mechanism that allows for selective removal of each.

Depending on the sequence detection modality used to read the homopolymer bit data strand, appropriate dNTP analog modifications can be designed that result in nucleotides without or with a lesion. For readout methods that rely on sequencing by synthesis (SBS), all modifications to purines or pyrimidines must be removed after homopolymer synthesis but before SBS readout. For readout methods that rely on current modulation during polymer translocation through one or more nanopores, modifications to purines and pyrimidines that are easily distinguishable from each other are desired. In some embodiments, modifications are made to purine and pyrimidine bases that modulate the polymerase kinetics during homopolymer synthesis. These "rate modulating" modifications can also act as current modulators for nanopore detection or can be removed for SBS detection.

Regardless of the mechanism of activation, it is important to control the length of the free-running homopolymer synthesis. Ideally, homopolymers of 2-4 nucleotides are desired. In the presence of natural nucleotides, TdT has a tendency to form homopolymers based on the nucleobases corresponding to the Km of the individual nucleotides (A>T>G>C). As pointed out by Lee HR et al (2018), it is difficult to limit homopolymer growth without increasing the deletion frequency. The authors reported about 66% deletion errors in an attempt to limit homopolymer growth to 2-3 nucleotides. Although TdT is reported to operate in a dispersive manner resulting in a Poisson distribution of homopolymer lengths in free-running synthesis, in practice, it is difficult to drive complete conversion of the initiator nucleic acid during homopolymer synthesis without long enzymatic extension reaction times. Too short a free-running homopolymer synthesis reaction time results in bit deletion errors, as reported above. Too long a homopolymer synthesis reaction time results in excessively long homopolymer formation and low efficient data density. One solution is to use modified nucleotide analogs that modulate the rate of incorporation of multiple bases but do not require removal at every step, thus allowing complete conversion of the starting nucleic acid, control of the length of the homopolymer formed, and maintaining the simple two-step cycle that is the subject of this patent. Ideally, the homopolymer tracts in the information polymer are greater than two nucleotides but four or fewer nucleotides in length.

Optionally, homopolymer length limiting modifications can be removed from all nucleotides at the end of synthesis, thus generating native DNA molecules suitable for SBS detection. Modifications that are chemically compatible with the decaging conditions described above are most desirable; they must be removable by chemical conditions that are orthogonal to those used for decaging. In some embodiments, the 3'-OH is caged with 3'-O-(2-nitrobenzyl), while the N6 of dA, the N4 of dC, the N2 of dG, and the O4 or N3 of dT are modified with non-terminating moieties that regulate multiple additions during free-running enzymatic homopolymer synthesis. Figure 24 shows a comparison of free-running homopolymer synthesis in the presence and absence of rate-regulating modifications. In some embodiments, the homopolymer synthesis rate-regulating modifications are the same for all four nucleotide analogs, while in other embodiments, the modifications are different for each of the dATP, dCTP, dGTP, and dTTP analogs. In some embodiments, the rate-limiting modifications remain in place during the entire synthesis.

Figures 25 and 26 show the results of two successive addition cycles of two differentially modified nucleotide analogs. In another embodiment, the nascent information polymer is exposed to periodic or alternating cycles having chemical conditions that result in partial or complete removal of the homopolymer rate-limiting modification. In some embodiments of the use of class 2 dNTP analogs, previously incorporated rate-controlling modifications are removed simultaneously with polymerase extension by including a mild reducing agent in alternating cycles of the enzymatic extension reaction mixture.

If a non-SBS method of readout (i.e., nanopore) is used, the homopolymer rate-modulating modification may be covalently attached using a non-cleavable linkage and left in place during readout. In some embodiments, the homopolymer rate-modulating modification may also be used to encode information during the readout process. In a preferred embodiment, the homopolymer synthesis rate-modulating analog is also designed to modulate the current during nanopore translocation and is further selected to provide two or more detectable and distinguishable levels of current blockage. In another embodiment, only a single type of nucleotide (i.e., A or C or G or T) is used in the information strand synthesis and the homopolymer bit is encoded by two or more differential current blockage-generating modifications.

Figures 27-30 show examples of solid-state nanopore detection of enzymatically synthesized homopolymers of peptide-modified dNTP analogs. A dTTP analog modified by a cleavable disulfide linkage to a 5 amino acid peptide (N-Ac-CYPEE) was used to generate fully modified molecules via free-running homopolymer synthesis greater than 100 nt in length. Translocation through a 2D silicon nitride 30 nm thick nanopore at 500 mV produced a large signal swing compared to unmodified dU homopolymer (courtesy of Goeppert LLC, Philadelphia, PA).

The following table describes six different classes of modified dNTP analogs useful for three different "delivery-free" homopolymer synthesis activation approaches and two different homopolymer-encoded nucleic acid memory strand readout technologies.

Examples of the four light-mediated decaging nucleotide analogs that make up Class I are shown below:

This class of analogs represents a 3'-O-caged dNTP that becomes a native unmodified dNTP upon light-mediated decaging. Once decaged, the rate of homopolymer formation does not differ from that of the native nucleotide, and the length of the homopolymer formed needs to be controlled by additives to the template-independent polymerase reaction formulation. In some embodiments, tetrahydropyranyl, 4-methoxy-tetrahydropyranyl, tetrahydrofuranyl, acetyl, methoxyacetyl, or phenoxyacetyl modifications are useful for thermally induced decaging of 3'-O blocked dNTP analogs. In some embodiments, 3'-O-analogs such as -O-CH ₂ -S-S-R are useful for electrochemically mediated decaging by reducing conditions. Class I dNTP analogs are characterized by nucleotides that yield unmodified homopolymers suitable for readout by either classical sequencing-by-synthesis or ratchet-style nanopores. In both cases, precision in the length of the homopolymer is not required, but precise detection of the transition between homopolymers is required.

Examples of four Class II photomediated de-caging nucleotide analogs with orthogonally removable peptide rate controlling modifications are shown below:

Peptide modifications for use with class II dNTP analogs are intended to be removed at the end of information polymer synthesis for subsequent interrogation by SBS-dependent sequencing methods or nanopore-mediated translocation, which are sensitive enough to detect the transition from one homopolymer type (A, C, G, T) to the next. Peptides suitable for use in this application slow down the rate of incorporation of modified nucleotide analogs to prevent long homopolymer formation prior to complete conversion of unmodified or modified strands of different composition. In some embodiments, the incorporation rate modulating modifications may consist of one, two, three or four different peptides for four nucleotide analogs. In some embodiments, the peptides are linked to the nucleotides via disulfide linkers that have a self-immolative scar that is cleavable under mild chemical conditions. In some embodiments, the peptides may consist of, but are not limited to, Ac-EECGY, Ac-EEGCGW, Ac-EEGCGGW, Ac-EC-pNA, Ac-CWEE, Ac-CYPEE, Ac-EEGCPPW, Ac-CPYEE, Ac-CPWEE, Ac-CWPEE. Many other peptide sequences and compositions are possible to one of skill in the art. Peptides with an overall anionic composition seem most suitable; peptides with a cationic composition accelerate the rate of nucleotide incorporation, as previously pointed out by Finn PS et al 2003. Peptides with covalent linkages to nucleotides other than via disulfides to cysteine amino acids are possible, so long as they maintain the ability to remove them from the nucleotides of completed homopolymeric DNA strands without leaving residual damage. Disulfide cleavage mediated self-immolative linkers, which are eliminated under mild conditions by the formation of thiolactones, are particularly useful.

The following are examples of four class II nucleotides with light-mediated decaging and orthogonally removable non-peptide homopolymer synthesis rate-regulating modifications:

Non-peptide modifications to N6 of adenine, N4 of cytidine, N2 of guanine, and N3 of thymine can act as incorporation rate modulators. In some embodiments, acetyl, diacetyl, isobutyryl, proprionyl, pivaloyl, benzoyl, cyclohexyl, and other organic modifiers are useful. Modifiers of this type are removed after information polymer synthesis, including but not limited to by ammonolysis. Class II dNTP analogs are characterized by nucleotides that yield unmodified homopolymers suitable for readout by either classical sequencing-by-synthesis or ratchet-style nanopores. In both cases, precision in the length of the homopolymer is not required, but precise detection of the transition between homopolymers is.

Below are examples of two Class III nucleotide analogs with an unmodified 3′-OH and a removable incorporation caging modification on the purine or pyrimidine:

In some embodiments, useful caging modifications to the 2-nitrobenzyl functionality are bulky modifications, including but not limited to peptides, cyclic peptides, PEG, branched PEG, star PEG, dendrimers, nanoparticles, etc. Class III dNTP analogs are also characterized by nucleotides that yield unmodified homopolymers suitable for readout by either classical sequencing-by-synthesis or ratchet-style nanopores. In both cases, precision in the length of the homopolymer is not required, but precise detection of the transition between homopolymers is.

An example of one Class IV nucleotide analog having an unmodified 3'-OH, a removable uptake blocker at a position other than the 3'-OH, and an orthogonally removable homopolymer synthesis rate modulator modification that is also not located at the 3'-OH is:

Class IV nucleotide analogs are designed to have one or more incorporation blocking modifications that cage the nucleotide from enzymatic incorporation until removed by light, heat or electrochemical means. Large bulky modifications that render the nucleotide analog inactive to polymerase incorporation are preferred. In some embodiments, the one or more modifications are derivatives of 2-nitrobenzyl that can be removed by photolysis. Removal of the caging modification in every cycle leaves the incorporation rate modulating modification that is removed by chemical methods that are orthogonal to the methods used to de-cage the nucleotide analog upon completion of the information polymer synthesis. In a preferred embodiment, the incorporation blocking modification is covalently linked to the incorporation rate modulating modification. In some embodiments, a caging modification to the 2-nitrobenzyl functionality is useful, which is a bulky modification and acts as a steric blocking agent. Examples of modifications that can act as steric blocking agents are, but are not limited to, peptides, proteins, peptoids, PEG, branched PEG, star PEG, dendrimers or nanoparticles.

Examples of Class V pyrimidine nucleotides having a 3'-O-caging modification and a non-removable base modification are:

If nanopore detection by direct translocation is desired as the readout method, the nucleotide is dually modified with a suitable removable 3'-O-caging group and a covalent non-removable modification that acts as a block current modulator during direct translocation through a non-indexed nanopore. Suitable modifications can be peptides or non-peptides. The ideal embodiment of a block current modulating group is the smallest possible modification that produces the most unique signal in the shortest possible homopolymer stretch. The key innovation of this class of dNTP analogs is that only one type of modified nucleotide is required, since the homopolymer "sequence" is encoded by the sequence of two or more current modulating modifications, not the nucleotide itself.

Below are examples of Class VI nucleotide analogs that contain an unmodified 3'-OH, a removable uptake blocking (caging) modification, and two or more different types of non-removable current modulating modifications useful for detection by direct nanopore sequencing. The key innovation of Class VI dNTP analogs is that only one type of modified nucleotide is required, since the homopolymer "sequence" is encoded by the sequence of two or more current modulating modifications, rather than the nucleotide itself. The encoding is not limited to base 2 or base 4, but is limited only by the number of current modulating modifications that can be made to a single purine or pyrimidine nucleotide.

In some embodiments, the entrapment blocking (caging) modification is removable by either light, heat or electrochemical means, while the two or more nanopore sensing elements are non-removable and survive repeated exposure to the de-caging conditions used in every cycle during homopolymer chain synthesis. The caging modification to 2-nitrobenzyl that acts as a steric blocking agent is a bulky modification such as, but not limited to, a peptide, protein, peptoid, PEG, branched PEG, star PEG, dendrimer or nanoparticle.

Example 1
N ⁶ -benzoyl-deoxyadenosine triphosphate was prepared by adding N ⁶ -benzoyl-2'-deoxyadenosine (0.055 g, 0.16 mmol) to a vial under dry N ₂ and trimethyl phosphate (0.435 mL) was added. To the resulting solution was added tributylamine (0.077 mL, 0.32 mmol) and the reaction mixture was flushed with dry N ₂ for 30 minutes while maintaining at -5°C. To this vial was added anhydrous phosphorus oxychloride (0.018 mL, 0.19 mmol) via syringe and the reaction mixture was stirred at -5°C for 3 minutes. A second aliquot of anhydrous phosphorus oxychloride (0.009 mL, 0.10 mmol) was added via syringe and the reaction mixture was stirred at -5°C for 8 minutes. To a second vial, tributylamine pyrophosphate (0.075 g, 0.14 mmol) was added, flushed with dry _N2 , anhydrous acetonitrile (0.609 mL) was added, followed by tributylamine (0.231 mL, 0.97 mmol). The prepared tributylamine pyrophosphate mixture was cooled to -20°C, added to the reaction mixture, and allowed to react for 10 minutes. The reaction was quenched by dropwise addition of _H2O (4.35 mL). The contents of the flask were combined with 0.87 mL of H2O and extracted with dichloromethane (3 x 150 mL). The aqueous phase was adjusted to pH 6.5 with concentrated _NH4OH and stirred at 4°C for 12 hours. The mixture was transferred to a 250 mL round bottom flask with 50 mL of water and concentrated under reduced pressure. The residue was dissolved in 40 mL water and purified via ion exchange chromatography (AKTA FPLC, Fractogel DEAE 48 mL column volume, step gradient 0->70% TEAB in water, pH 7.5). Fractions containing the desired product were pooled, concentrated under reduced pressure, concentration by A260, and residual triethylammonium bicarbonate was removed via repeated concentration from water to dryness (5 x 50 mL) to give N ^-benzoyl -2'-deoxyadenosine triphosphate.

Controlled synthesis of homopolymeric tracts composed of modified nucleotides by nucleotidyl transferase TdT was carried out in the following manner: Stock solutions of 1 mM each of deoxyadenosine triphosphate (TriLink Biosciences) and N ⁶ -benzoyl-deoxyadenosine triphosphate were prepared in H ₂ O.

0.5 μL (500 pmoles) of each of the different triphosphates was separately combined with 1.5 μL (30 U) of commercial TdT (Thermo Scientific), 0.5 μL (50 pmoles) of 5'-TAATAATAATTTTT-3' (IDT), 2 μL of commercial TdT Rxn buffer (Thermo Scientific - 1 M potassium cacodylate, 0.125 M Tris, 0.05% (v/v) Triton X100, 5 mM _CoCl2 (pH 7.2 at 25°C)) and 4.5 μL of H2O. Reactions were incubated at 37°C. 30 μL aliquots were removed after 1, 5 and 15 min and each quenched with 20 μL 5 mM EDTA. Each sample was dried under vacuum and reconstituted in 100 μL H2O. 10 μL of each time point was mixed with 10 μL of denaturing loading buffer (100% formamide and 0.1% Orange G) and applied to the wells of a 1 mm x 20 cm x 14 cm 20% polyacrylamide gel. After 3.5 hours of electrophoresis at 400 V, bands were visualized by Sybr Gold (Thermo Scientific) and photographed under UV illumination (UV-blocking Wratten 2A filter 405 nm cutoff, UVP, LLC).

For synthesis of multiple homopolymer tracts, initiator oligonucleotides can be attached to beads to allow multiple rounds of enzymatic synthesis incorporating removal of previous reactants and washes. 5'-biotin-TAATAATAATTTTT-3' (IDT) can be incubated with streptavidin-coated magnetic sepharose microbeads (GE Healthcare Life Sciences). Oligonucleotide-loaded beads can be prepared by removing an aliquot of bead slurry and transferring to a filter cup. The beads can then be washed 5 times with 1x PBS (using 2x volume of bead slurry) vortexing, each rinse can be spun down. 1/2 bead slurry volume of 1x PBS can then be added and biotinylated oligonucleotides can be spiked at 1/10 of the published bead binding capacity. The mixture can be incubated at 37C for 2 hours with vortexing every 30 minutes. After 2 hours, a small amount of supernatant can be removed and the A260 can be measured for any unbound oligonucleotide. When the A260 indicates less than 10% remaining oligonucleotide, the beads can be washed five times with MQ water. The washed beads can be brought to the desired concentration in MQ water.

Homopolymer synthesis can be carried out as above using 2-10x molar equivalents of the desired dNTPs relative to the bead-bound oligonucleotides. The reaction mixture containing beads, dNTPs, TdT and buffer can be incubated at 37°C for 15 minutes. The reaction can be stopped with 10 μl EDTA and rinsed three times with water. A new cycle of homopolymer synthesis can be initiated by adding fresh TdT enzyme, dNTPs, buffer and incubated at 37°C for 15 minutes. After stopping the reaction with EDTA and rinsing three times with water, the cycle of homopolymer synthesis can be repeated as many times as desired. After the final EDTA quench and three rinses with water, the support-bound alternating homopolymer can be cleaved from the solid support using 100 μl concentrated ammonium and the supernatant can be dried by Gen-vac and then stored at -20°C until ready to be analyzed using polyacrylamide gels as above.

In another experiment, as shown in Figure 12, the two-letter message "MA" was converted to binary and then to base-2 nucleotide code. Each letter symbol was converted to a corresponding number from 1 to 26 (A→1, ...Z→26). Each number was then converted to a 6-bit binary number by appending two zeros to the normal binary representation of 1 to 26 (e.g., "M"=001101;"A"=000001). Each bit of the binary representation was converted to a base-2 nucleotide representation according to the following table:

Thus, "MA" is converted to 001101 000001, which is then converted to the single nucleotide string ACTGAGACACAG, which is synthesized as A _n C _n T _n G _n A _n G _n A _n C _n A _n C _n A n _{G n ,} where each nucleotide is synthesized as a homopolymer of variable length.

Synthesis of 12-bit homopolymer encoded nucleic acids was performed using a 5'-biotinylated 39 nt long oligonucleotide initiator: 5'biotin-CAGGTCCTAUC GATATC TGTGAGCTTAATGTCCTTATGT-3' bound to 34 um streptavidin-sepharose beads (GE Healthcare) at approximately 20 pmol/ul of beads.

This oligonucleotide contains two features to release the final product from the solid support used during synthesis: 1) a single deoxyuridine residue that allows cleavage with the USER enzyme system (New England Biolabs), and 2) an Eco R V endonuclease restriction site.

Starting with approximately 2 nmol of bead-bound initiator, homopolymers of each variable length were enzymatically synthesized using TdT and one of four modified nucleotide triphosphates. Each reaction was carried out in a total volume of 750 ul containing 40-100 uM modified dNTPs (40 uM-A; 100 uM-C; 50 uM-T; 100 uM-G), 20 U TdT (Thermo-Fisher Scientific), and 1x TdT buffer (Thermo-Fisher Scientific) with incubation at 37°C for 2.5-20 min. After each enzymatic extension step, the reaction was quenched by adding 500 ul of 250 mM EDTA in 10 mM Tris buffer (pH 6.8). The beads were collected by centrifugation at 10,000×g and removal of the supernatant. FIG. 13 shows a PAGE analysis of each cycle of enzymatic synthesis of a 12-bit homopolymeric data strand. Each lane begins with "N" indicating unreacted 39 nt initiator and is marked with the cycle number. The black arrow indicates the size marker of the 60 nt oligonucleotide.

After removal of the full-length data strand from the solid support, NGS library preparation was performed using the ACCEL-NGS® 1S PLUS DNA LIBRARY KIT (Swift Bioscience) according to the manufacturer's instructions and subsequently sequenced using an Illumina MiSeq System. Figures 14A-L show histograms of the observed base composition of each of the 12 nucleotide additions (as labeled) generated during enzymatic synthesis.

Example 2
Procedures for the Synthesis of Class I - Purine and Pyrimidine dNTP Analogs Scheme for the Synthesis of Class I dNTP Analogs Nitrobenzyl-Adenosine
Nitrobenzyl-cytosine
Nitrobenzyl-guanosine
Nitrobenzyl-thymidine

Example 3
Detailed procedure for 3'-O-nitrobenzyldeoxyadenosine:
9-[β-D-5'-hydroxy-2'-deoxyribofuranosyl]-6-chloropurine (1.00 g, 3.69 mmol) and imidazole (554 mg, 8.12 mmol) were dissolved in anhydrous dimethyl formate (18 mL) followed by the addition of tert-butyldimethylsilyl chloride (611 mg, 3.93 mmol). The reaction mixture was stirred at room temperature under argon for 20 h. The dried residue was impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 2:1) to give 9-[β-D-5'-O-(tert-butyldimethylsilyl)-2'-deoxyribofuranosyl]-6-chloropurine.

9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloropurine (1.73 g, 4.48 mmol) was dissolved in anhydrous dichloromethane (135 mL). Tetrabutylammonium bromide (722 mg, 2.24 mmol), 2-nitrobenzyl bromide (2.41 g, 11.2 mmol) and 40% aqueous sodium hydroxide (65 mL) were added to the pre-made solution. The reaction mixture was stirred at room temperature for 1 hour and diluted with ethyl acetate (300 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (125 mL×2). The combined organic layers were dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica gel and then purified by flash column chromatography (hexane/ethyl acetate, 2:1) to give 9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)-2′-deoxyribofuranosyl]-6-chloropurine.

9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)-2′-deoxyribofuranosyl]-6-chloropurine (2.22 g, 3.83 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) and cooled to 0° C. Then, 1.0 M tetrabutylammonium fluoride solution in tetrahydrofuran (4.20 mL, 4.20 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 h. After drying the reaction mixture, the residue was dissolved in dioxane and 7N ammonia in ethanol (40 mL). The reaction mixture was stirred at 90° C. for 18 h in a sealed round bottom. The reaction mixture was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 20:1) to give 3′-O-(2-nitrobenzyl)-2′-deoxyadenosine.

3'-O-(2-nitrobenzyl)-2'-deoxyadenosine (15 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added in one portion as a solid. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

Example 4
Detailed procedure for 3'-O-nitrobenzyldeoxycytidine:
3',5'-Di-O-(tert-butyldimethylsilyl)-2'-deoxyuridine (1.00 g, 2.12 mmol) was dissolved in anhydrous acetonitrile (90 mL) and cooled to 0° C. under argon. Phosphoryl trichloride (1.49 mL, 2.12 mmol) was added dropwise over 2 minutes. After 10 minutes, triethylamine (11.1 mL, 79.7 mmol) was added dropwise over 3 minutes. After 15 minutes, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0° C. and triazole (4.40 g, 63.7 mmol) was added as a solid in one portion. A precipitate was observed and the suspension was stirred for 30 minutes. After stirring at room temperature for 2 hours, the reaction mixture was concentrated to dryness. The residue was dissolved in dichloromethane (30 mL) and washed with a saturated solution of sodium bicarbonate (25 mL×2), brine (25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was dissolved in dichloromethane, followed by the addition of allyl alcohol (2.00 mL, 29.4 mmol) and triethylamine (2.67 mL, 18.9 mmol). The reaction mixture was stirred at 0° C. for 15 min. DBU (0.33 mL, 2.17 mmol) was added and stirred at room temperature for 6 h. The reaction mixture was diluted with dichloromethane (17 mL) and washed with brine (15 mL). The organic layer was dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 4:1) to give 4-O-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyuridine.

4-O-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyuridine (3.37 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 4-O-allyl-2'-deoxyuridine.

4-O-allyl-2'-deoxyuridine (1.18 g, 4.40 mmol) was dissolved in anhydrous pyridine (37 mL) followed by the addition of tert-butyldimethylsilyl chloride (815 mg, 5.41 mmol) under argon. The reaction mixture was stirred at room temperature for 20 h. After concentration, the residue was dissolved in dichloromethane and impregnated onto silica. The crude product was purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 4-O-allyl-5'-O-(tert-butyldimethylsilyl)-2'-deoxyuridine.

To a mixture of 4-O-allyl-5'-O-(tert-butyldimethylsilyl)-2'-deoxyuridine (1.28 g, 3.35 mmol), tetrabutylammonium hydroxide (1.5 mL, 55-60% in water) and sodium iodide (50.0 mg, 0.335 mmol) in dichloromethane/water (20 mL, 1:1) was added 1.0 M sodium hydroxide solution (10 mL) under argon. The reaction mixture was stirred at room temperature for 10 minutes, after which 2-nitrobenzyl bromide (1.45 g, 6.70 mmol) in 10 mL dichloromethane was added over 5 minutes. After stirring at room temperature for 7 hours, the reaction mixture was diluted with dichloromethane (150 mL). The organic layer was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 1:1) to give 4-O-allyl-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxyuridine.

4-O-allyl-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxyuridine (1.55, 3.00 mmol) was dissolved in 7N ammonia in ethanol (55 mL) and stirred in a sealed round bottom for 20 hours at 55° C. The reaction mixture was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 20:1) to give 5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (2.22 g, 3.83 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) and cooled to 0°C. Then, 1.0 M tetrabutylammonium fluoride solution in tetrahydrofuran (4.20 mL, 4.20 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 8:2) to obtain 3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

3'-O-(2-nitrobenzyl)-2'-deoxycytidine (14 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added in one portion as a solid. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 5)
Detailed procedure for 3'-O-nitrobenzyldeoxyguanosine:
2-Amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]purine (1.00 g, 3.50 mmol) and imidazole (715 mg, 10.50 mmol) were dissolved in anhydrous dimethyl formate (18 mL) followed by the addition of tert-butyldimethylsilyl chloride (686 mg, 4.60 mmol). The reaction mixture was stirred at room temperature under argon for 12 h. The dried residue was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 2-amino-6-chloro-9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]purine.

2-Amino-6-chloro-9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]purine (1.19 g, 3.00 mmol) was dissolved in anhydrous tetrahydrofuran (8.0 mL) and then N,N-dimethylformamide dimethyl acetal (3.10 mL, 18.0 mmol) was added at room temperature. The reaction mixture was stirred at 40° C. for 3 hours. The reaction mixture was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 6-chloro-N ² -[(dimethylaminomethylene)amino]-9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]purine.

6-Chloro-N ² -[(dimethylaminomethylene)amino]-9-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]purine (1.09 g, 2.40 mmol) was dissolved in anhydrous acetonitrile (3.5 mL) followed by the addition of sodium hydride powder (60%) in mineral oil (122 mg, 4.80 mmol) at 0° C. After stirring for 1 h at room temperature, a solution of 2-nitrobenzyl bromide (1.04 g, 4.80 mmol) in anhydrous acetonitrile (1.5 mL) was added. After stirring for 2 h at room temperature, the reaction mixture was filtered. The filtrate was dried and the resulting residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with saturated sodium bicarbonate solution (50 mL), brine (50 mL) and dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate 4:6) to give 6-chloro-N ² -[(dimethylaminomethylene)amino]-9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)-2′-deoxyribofuranosyl]purine.

6-Chloro-N ² -[(dimethylaminomethylene)amino]-9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)-2′-deoxyribofuranosyl]purine (1.02 g, 1.73 mmol) was dissolved in dimethyl formate anhydride (15 mL) followed by the addition of cesium acetate (996 mg, 5.19 mmol), 1,4-diazabicyclo[2.2.2]octane (194 mg, 1.73 mmol) and triethylamine (0.72 mL, 5.19 mmol) under argon. The reaction mixture was stirred at room temperature for 18 hours. Acetic anhydride (5 mL) was added and stirred for 0.5 hours. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (dichloromethane/methanol, 20:1) to give 5'-O-(tert-butyldimethylsilyl)-N ² -[(dimethylamino)methylene]-3'-O-(2-nitrobenzyl)-2'-deoxyguanosine.

5'-O-(tert-butyldimethylsilyl)-N ² -[(dimethylamino)methylene]-3'-O-(2-nitrobenzyl)-2'-deoxyguanosine (732 mg, 1.28 mmol) was dissolved in anhydrous tetrahydrofuran (8 mL) under argon and cooled to 0°C. Then, 1.0 M tetrabutylammonium fluoride solution in tetrahydrofuran (2.56 mL, 2.56 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL x 2). The organic layers were combined and dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 10:1) to give N ² -[(dimethylamino)methylene]-3'-O-(2-nitrobenzyl)-2'-deoxyguanosine.

N ² -[(dimethylamino)methylene]-3'-O-(2-nitrobenzyl)-2'-deoxyguanosine (17 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight on high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added as a solid in one portion. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

Example 6
Detailed procedure for 3'-O-nitrobenzyldeoxythymidine:
Thymidine (2.50 g, 10.3 mmol) was suspended in dimethylformamide (60 mL) at room temperature under argon. To this suspension was added imidazole (4.22 g, 61.9 mmol) and tert-butylchlorodimethylsilane (4.66 g, 30.1 mmol). After stirring for 2 h, the reaction mixture was quenched with methanol (8 mL) and diluted with ethyl acetate (200 mL). The organic layer was washed with water (100 mL x 2), a saturated solution of sodium bicarbonate (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 8:2) to give 3',5'-di-O-(tert-butyldimethylsilyl)thymidine.

3',5'-Di-O-(tert-butyldimethylsilyl)thymidine (4.34 g, 9.22 mmol) and dimethyl-4-aminopyridine (1.12 g, 9.22 mmol) were dissolved in anhydrous dichloromethane (140 mL). Triethylamine (5.14 mL, 36.9 mmol) was added and the reaction mixture was cooled to 0° C. Benzyl chloride (3.21 mL, 27.7 mmol) was added dropwise and warped up to room temperature. After stirring for 14 hours, a saturated solution of sodium bicarbonate (80 mL) was added and the layers were separated. The aqueous layer was extracted with dichloromethane (200 mL×2). The combined organic layers were washed with water (300 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 8:2) to give 3-N-benzoyl-3'5'-di-O-(tert-butyldimethylsilyl)thymidine.

3-N-benzoyl-3'5'-di-O-(tert-butyldimethylsilyl)thymidine (3.03 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 3-N-benzoylthymidine.

3-N-benzoylthymidine (XX g, 4.40 mmol) was dissolved in anhydrous pyridine (37 mL) followed by the addition of tert-butyldimethylsilyl chloride (815 mg, 5.41 mmol) under argon. The reaction mixture was stirred at room temperature for 20 h. After concentration, the residue was dissolved in dichloromethane and impregnated onto silica. The crude product was purified by flash column chromatography (dichloromethane/methanol, 9:1) to give 3-N-benzoyl-5'-O-(tert-butyldimethylsilyl)thymidine.

To 3-N-benzoyl-5'-O-(tert-butyldimethylsilyl)thymidine (1.18 g, 2.56 mmol) was added an aqueous solution of tetrabutylammonium hydroxide (10 mL, 60%), followed by sodium iodide (76.7 mg, 0.51 mmol), dichloromethane (10 mL), water (10 mL), and an aqueous solution of 1 M sodium hydroxide (10 mL). This mixture was added dropwise to a solution of 2-nitrobenzyl bromide (718 mg, 3.32 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 6 hours, after which water (10 mL) was added. The aqueous layer was extracted with dichloromethane (50 mL x 3). The organic layers were combined, washed with brine (100 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the residue was dissolved in ethyl acetate, impregnated onto silica, and purified by flash column chromatography (hexane/ethyl acetate, 6:4) to give 3-N-benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)thymidine.

3-N-benzoyl-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)thymidine (1.24 g, 2.08 mmol) was dissolved in ethanol (15 mL) followed by the addition of 30% ammonium hydroxide solution (1.5 mL, 12.3 mmol). The reaction mixture was stirred at room temperature for 1 h, impregnated onto silica and purified by flash column chromatography (hexane/ethyl acetate, 6:4) to give 5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)thymidine.

5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)thymidine (681 mg, 1.39 mmol) was dissolved in anhydrous tetrahydrofuran (12 mL) under argon and cooled to 0°C. Then, 1.0 M tetrabutylammonium fluoride solution in tetrahydrofuran (2.78 mL, 2.78 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic layers were combined and dried over anhydrous sodium sulfate. The organic layer was impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 10:1) to give 3'-O-(2-nitrobenzyl)thymidine.

3'-O-(2-nitrobenzyl)thymidine (15 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 sec to the rxn mixture. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added in one portion as a solid. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

Example 6
Class II - Procedures for synthesizing purine and pyrimidine dNTP analogs.
Scheme for the synthesis of class II non-peptide dNTP analogs. Non-peptide precursor - adenosine
Non-peptide precursor - cytosine
Scheme for the synthesis of class II peptide-dNTP analogs Disulfide peptide precursor - Adenosine
Disulfide Peptide Precursor - Cytosine

(Example 7)
Detailed procedure for the synthesis of class II non-peptide dATP constructs:
9-[β-D-5′-O-(tert-butyldimethylsilyl)-3′-O-(2-nitrobenzyl)-2′-deoxyadenosine (0.24 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL) at room temperature under argon. Sodium hydride (48 mg, 1.21 mmol) was added in one portion. After stirring for 1 h, the reaction mixture was cooled to 0° C. and acyl chloride (3 eq, 0.723 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was poured into a cold solution of saturated sodium bicarbonate and dichloromethane. The layers were separated. The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (hexanes/ethyl acetate, 2:8) to give the desired product.

The N-substituted nucleoside (5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give the N-substituted 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine.

N-substituted 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine (1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added in one portion as a solid. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 8)
Detailed procedure for the synthesis of class II non-peptide dCTP constructs:
5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (524.3 mg, 1.10 mmol) and carboxylic acid (1.32 mmol) were dissolved in anhydrous dimethyl formate under argon. N,N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (hexane/ethyl acetate, 4:6) to give N-substituted 5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

N-substituted 5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (3.37 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give N-substituted 3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

N-substituted 3'-O-(2-nitrobenzyl)-2'-deoxycytidine (1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added in one portion as a solid. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 9)
Detailed procedure for the synthesis of peptide-dATP conjugates:
9-[β-D-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxyadenosine (524.3 mg, 1.10 mmol) and 4-(pyridin-2-yldisulfaneyl)butanoic acid (302.7 mg, 1.32 mmol) were dissolved in anhydrous dimethyl formate under argon. N,N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (hexane/ethyl acetate, 4:6) to give N-(4-(pyridin-2-yldisulfanyl)butanyryl)-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxyadenosine.

N-(4-(pyridin-2-yldisulfanyl)butanyl)-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxyadenosine (3.75 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give N-(4-(pyridin-2-yldisulfanyl)butanyl)-3'-O-(2-nitrobenzyl)-2'-deoxyadenosine.

N-(4-(pyridin-2-yldisulfanyl)butanyl)-3'-O-(2-nitrobenzyl)-2'-deoxyadenosine (23 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight on high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for a further 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 sec to the rxn mixture. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added as a solid in one portion. The mixture was stirred for 10 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 10)
Detailed procedure for the synthesis of peptide-dCTP conjugates:
5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (524.3 mg, 1.10 mmol) and 4-(pyridin-2-yldisulfanyl)butanoic acid (1.32 mmol) were dissolved in anhydrous dimethyl formate under argon. N,N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (hexane/ethyl acetate, 4:6) to give N-(4-(pyridin-2-yldisulfanyl)butanyl)-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

N-(4-(pyridin-2-yldisulfanyl)butanyl)-5'-O-(tert-butyldimethylsilyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (3.62 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated onto silica, and purified by flash column chromatography (dichloromethane/methanol, 9:1) to give N-(4-(pyridin-2-yldisulfanyl)butanyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine.

N-(4-(pyridin-2-yldisulfanyl)butanyl)-3'-O-(2-nitrobenzyl)-2'-deoxycytidine (22 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight on high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 sec. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added as a solid in one portion. The mixture was stirred for 10 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 11)
Class III - Procedures for synthesizing purine and pyrimidine dNTP analogs.
Scheme for the synthesis of class III dNTP analogs

Example 12
Detailed Procedure for Class III-Purine dNTP Analogs:
To a solution of phosgene (6.46 g, 2 eq, 65.3 mmol) in dry toluene (100 mL) at 23° C. was added (2-nitrophenyl)methanol (5.00 g, 1 eq, 32.6 mmol) in 20 mL dry THF. The reaction was stirred at 23° C. for 24 h. The reaction was then concentrated to dryness under vacuum with a trap of aqueous NaOH. The residual amber oil was used directly in the reaction without further purification.

To a solution of 9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-amine (12.2 g, 1.1 eq, 25.5 mmol) in dry DMF (100 mL) at 0° C. was added N,N-diisopropylethylamine (3.60 g, 4.9 mL, 1.2 eq, 27.8 mmol). The reaction was stirred for 30 minutes and then 2-nitrobenzyl carbonochloridate (5.00 g, 1 eq, 23.2 mmol) was added dropwise slowly over 30 minutes while maintaining the temperature below 5° C. The reaction was then allowed to warm to room temperature and stirred overnight. The reaction was poured into a cooled solution of 5% Na2CO3 and EtOAc. The EtOAc layer was dried over sodium sulfate and then concentrated to dryness. The crude product was chromatographed on silica gel using hexane/EtOAc mixtures to give the purified product, which was used in the next reaction.

2-Nitrobenzyl (9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)carbamate (5.00 g, 1 eq, 7.59 mmol) was dissolved in THF at room temperature and then cooled to OC under a blanket of dry argon. To the mixture was then added tetrabutylammonium fluoride (4.96 g, 2.5 eq, 19.0 mmol). The mixture was stirred at 0 C for 2 h and then warmed to 23 C for 1 h. The solution was poured into a cold solution of 10% NaHCO3 and extracted with DCM. The DCM layer was concentrated and the crude product was purified on silica gel eluting with 5-50% DCM/MeOH to give the product suitable for triphosphorylation.

2-Nitrobenzyl (9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)carbamate (28 mg, 1 eq, 65 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL dry pyridine and cooled in an ice bath under argon. A first aliquot of phosphoryl trichloride (30 mg, 18 μL, 3 eq, 0.20 mmol) was added. After 5 min, a second aliquot of 10 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (0.23 g, 4 eq, 0.26 mmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 sec to the reaction mixture at rxn t=35 min. Immediately, pre-weighed N1,N1,N8,N8-tetramethyl-naphthalene-1,8-diamine (56 mg, 4 eq, 0.26 mmol) was added as a solid in one portion. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation.

Example 13
Detailed Procedure for Class III-Pyrimidine dNTP Analogs:
To a solution of 4-amino-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)-methyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (9.30 g, 1.1 eq, 20.4 mmol) in dry DMF (100 mL) at 0° C. was added N,N-diisopropylethylamine (2.88 g, 3.9 mL, 1.2 eq, 22.3 mmol). The reaction was stirred for 30 minutes and then 2-nitrobenzyl carbonochloridate (4.00 g, 1 eq, 18.6 mmol) was added dropwise slowly over 30 minutes while maintaining the temperature below 5° C. The reaction was then allowed to warm to room temperature and stirred overnight. The reaction was poured into a cooled solution of 5% Na2CO3 and EtOAc. The EtOAc layer was dried over sodium sulfate and then concentrated to dryness. The crude product was chromatographed on silica gel using hexane/EtOAc mixtures to give the purified product, which was used in the next reaction.

2-Nitrobenzyl (1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (5.00 g, 1 eq, 7.88 mmol) was dissolved in 25 mL THF at room temperature and then cooled to OC under a blanket of dry argon. To the mixture was then added tetrabutylammonium fluoride (5.15 g, 2.5 eq, 19.7 mmol). The mixture was stirred at 0 C for 2 h and then warmed to 23 C for 1 h. The solution was poured into a cold solution of 10% NaHCO3 and extracted with DCM. The DCM layer was concentrated and the crude product was purified on silica gel eluting with 5-50% DCM/MeOH to give the product suitable for triphosphorylation.

2-Nitrobenzyl (1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (35.0 mg, 1 eq, 86.1 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL dry pyridine and cooled in an ice bath under argon. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 min, a second aliquot of 10 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 sec to the reaction mixture at rxn t=35 min. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 eq, 345 μmol) was added as a solid in one portion. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred for 30 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation.

(Example 14)
Procedure for synthesizing class IV-dCTP analogs.
Scheme for the synthesis of class IV dCTP analogs
Detailed Procedure for Class IV dCTP Analogs:

Methyl 3,4,5-trihydroxybenzoate (10 g, 1 eq, 54 mmol) was dissolved in 50 mL of acetone. Sodium iodide (0.81 g, 0.1 eq, 5.4 mmol) and potassium carbonate (38 g, 5 eq, 270 mmol) were added as solids at ambient temperature. 1-(chloromethyl)-2-nitrobenzene (34 g, 3.6 eq, 0.20 mol) was added dropwise over 10 minutes as a solution in 40 mL of acetone. The mixture was stirred for 1 hour and then heated to 50° C. for 6 hours. The mixture was cooled to ambient temperature and the bulk of the solvent was removed on a rotary evaporator. The residue was suspended in 200 mL of EtOAc, which was washed successively with 200 mL portions of water and saturated aqueous NaCl. The EtOAc layer was dried over sodium sulfate and evaporated. The crude product was chromatographed on silica using hexane/EtOAc mixtures to give a purified product which could be used in the next reaction.

Methyl 3,4,5-tris((2-nitrobenzyl)oxy)benzoate (25 g, 1 eq, 42 mmol) was dissolved in 300 mL of THF. Aqueous 2 M NaOH (105 mL, 210 mmol) was added and the mixture was stirred at ambient temperature for 18 h. The bulk of the THF was removed on a rotary evaporator and the residue was slowly acidified with 6 M HCl to a pH of 1 or less. The resulting solid was filtered, washed thoroughly with water, and dried on the filter funnel for 5 h and then dried under high vacuum for 18 h. The product was carried forward to the next reaction without further purification.

4-Amino-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (12 g, 1 eq, 26 mmol) and 3,4,5-tris((2-nitrobenzyl)oxy)benzoic acid (15 g, 1 eq, 26 mmol) were dissolved in 50 mL of dry DMF at ambient temperature under an argon atmosphere. N-Ethyl-N-isopropylpropan-2-amine (5.1 g, 6.8 mL, 1.5 eq, 39 mmol) was added followed by a solution of 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (V) (12 g, 1.2 eq, 31 mmol) in 10 mL of dry DMF dropwise over 5 min at ambient temperature. The mixture was stirred at ambient temperature for 18 h. The mixture was dissolved in 300 mL of EtOAc, which was washed successively with 200 mL portions of water (2×) and saturated aqueous NaCl. The EtOAc was dried over sodium sulfate and evaporated first on a rotary evaporator and then under high vacuum for 18 h. The residue was chromatographed on silica using a mixture of dichloromethane and methanol to give the desired product as a colorless foam.

N-(1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3,4,5-tris((2-nitrobenzyl)oxy)benzamide (5 g, 1 eq, 5 mmol) was dissolved in 25.0 mL of dry THF at ambient temperature under argon. Triethylamine (4 g, 6 mL, 8 eq, 4e+1 mmol) was added rapidly followed by triethylammonium fluoride dihydrofluoride (5 g, 5 mL, 6 eq, 3e+1 mmol), also at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel (20 g) was added and the mixture was evaporated on a rotary evaporator to a fine powder then loaded onto a 100 g silica column and eluted with a mixture of dichloromethane and methanol to give the nucleoside as a slightly yellow foam.

N-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3,4,5-tris((2-nitrobenzyl)oxy)benzamide (30 mg, 1 eq, 38 μmol) was coevaporated with pyridine (1 mL x 3) and dried overnight on high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 min, a second aliquot of 5 uL was added. The mixture was stirred for a further 30 min. A solution of tetrabutylammonium dihydrogen phosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 seconds to the rxn mixture. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added as a solid in one portion. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

(Example 15)
Procedures for synthesizing Class V peptides and non-peptide analogs.
Scheme for the synthesis of peptide-thymidine dNTP conjugates
Scheme for the synthesis of non-peptide-thymidine dNTP conjugates

(Example 16)
Detailed procedure for peptide-dTTP analogs:
4-(Bromomethyl)benzenethiol (5.00 g, 1 eq, 24.6 mmol) was dissolved in methanol (50 mL) and cooled to 0° C. To the mixture was then added 1,2-di(pyridin-2-yl)disulfane (5.42 g, 1 eq, 24.6 mmol) and stirred at 0° C. for 18 h. The reaction was then concentrated directly and purified on silica get eluting with hexanes/ethyl acetate (0-100% EtOAc) to give the product as a white solid which was used directly in the next reaction.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 50 mL DMF and then cooled to 0 C. The reaction was stirred at 0 C for 30 minutes and then sodium hydride (306 mg, 1.2 eq, 12.7 mmol) was added. The reaction was then stirred at 0 C for an additional 30 minutes and then warmed to 23 C. To the mixture was then added 2-((4-(bromomethyl)phenyl)-disulfanyl)-pyridine (3.32 g, 1 eq, 10.6 mmol) and stirring was continued at 23 C for an additional 2 hours. The reaction was then poured into a cold solution of 10% NaHCO3 and DCM. The DCM layer was separated, dried over sodium sulfate and concentrated to dryness. The mixture was then purified on silica gel eluting with 0-20% DCM/methanol to give the desired product.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)-methyl)tetrahydrofuran-2-yl)-5-methyl-3-(4-(pyridin-2-yldisulfanyl)benzyl)pyrimidine-2,4(1H,3H)-dione (2.00 g, 1 eq, 2.85 mmol) was dissolved in THF and cooled to 0 C. To the mixture was then added tetrabutylammonium fluoride (2.23 mg, 3 eq, 8.55 mmol) at 0 C. The reaction was continued to stir at 0 C for 2 h and then warmed to 23 C for an additional 1 h. The reaction was then cooled to 0 C again and added to a pre-cooled solution of 10% NaHCO3 and DCM at 0 C. The DCM layer was then separated, dried over sodium sulfate, concentrated and purified on silica gel eluting with 5-50% DCM/methanol to give the pure product.

1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methyl-3-(4-(pyridin-2-yldisulfanyl)benzyl)pyrimidine-2,4(1H,3H)-dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 20 mL of THF at 23 C. To the mixture was then added triethylamine (1.07 g, 1.5 mL, 1 eq, 10.6 mmol) and cooled to 0 C. To the mixture was then added TBS-Cl (1.59 g, 1 eq, 10.6 mmol) and stirring was continued at 0 C for an additional 2 h. The mixture was then added to a pre-cooled mixture of 10% aqueous NaCl and DCM. The DCM layer was dried over sodium sulfate and concentrated to dryness to give an amber oil. The crude product was then purified on silica gel eluting with 5-50% DCM/methanol to give the pure product.

1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-(4-(pyridin-2-yldisulfanyl)benzyl)pyrimidine-2,4(1H,3H)-dione (2.00 g, 1 eq, 3.40 mmol) was dissolved in 20 mL DMF and then cooled to 0 C. To the mixture was then added sodium hydride (98.0 mg, 1.2 eq, 4.08 mmol) and stirring was continued at 0 C for an additional 30 min. To the reaction was then added 1-(bromomethyl)-2-nitrobenzene (735 mg, 1 eq, 3.40 mmol) and stirring was continued at 0 C for an additional 1 h. The reaction was then added to a pre-cooled mixture of 10% NaCl and EtOAc. The EtOAc layer was separated, dried over sodium sulfate and concentrated to dryness. The crude product was purified on silica gel eluting with 0-50 hexanes/EtOAc to give the desired product.

1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-nitrobenzyl)oxy)tetrahydrofuran-2-yl)-5-methyl-3-(4-(pyridin-2-yldisulfanyl)benzyl)pyrimidine-2,4(1H,3H)-dione (1.00 g, 1 eq, 1.38 mmol) was dissolved in THF and cooled to 0 C. To the reaction was then added TBAF (362 mg, 1 eq, 1.38 mmol) at 0 C and stirred at 0 C for 1 h then warmed to room temperature over 2 h. The mixture was then poured into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was separated and dried over sodium sulfate. The crude product was purified on silica gel eluting with 5-25% DCM/methanol to give the desired product.

4-(pyridin-2-yl)benzyl (1-((2R,4S,5R)-5-(hydroxymethyl)-4-((2-nitrobenzyl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (35.0 mg, 1 eq, 61.0 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 min, a second aliquot of 10 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 seconds to the reaction mixture at rxn t=35 minutes. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 eq, 345 μmol) was added in one portion as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation.

(Example 17)
Detailed Procedure for Non-peptide-dTTP Analogues:
1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)-oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 50 mL DMF and then cooled to 0 C. The reaction was stirred at 0 C for 30 min and then sodium hydride (306 mg, 1.2 eq, 12.7 mmol) was added. The reaction was stirred at 0 C for an additional 30 min and then warmed to 23 C. To the mixture was then added (bromomethyl)benzene (1.82 g, 1 eq, 10.6 mmol) and stirring was continued at 23 C for an additional 2 h. The reaction was then poured into a cold solution of 10% NaHCO3 and DCM. The DCM layer was separated, dried over sodium sulfate and concentrated to dryness. The mixture was then purified on silica gel eluting with 0-20% DCM/methanol to give the desired product.

3-Benzyl-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)-methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (4.00 g, 1 eq, 7.13 mmol) was dissolved in THF and cooled to 0 C. To the mixture was then added tetrabutylammonium fluoride (3.72 g, 2 eq, 14.26 mmol) at 0 C. The reaction was continued to stir at 0 C for 2 h and then warmed to 23 C for an additional 1 h. The reaction was then cooled again to 0 C and added to a pre-cooled solution of 10% NaHCO3 and DCM at 0 C. The DCM layer was then separated, dried over sodium sulfate, concentrated and purified on silica gel eluting with 5-50% DCM/methanol to give the pure product.

3-Benzyl-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.00 g, 1 eq, 3.01 mmol) was dissolved in 20 mL of THF at 23 C. To the mixture was then added triethylamine (304 mg, 0.42 mL, 1 eq, 3.01 mmol) and cooled to 0 C. To the mixture was then added TBS-Cl (453 mg, 1 eq, 3.01 mmol) and stirring was continued at 0 C for an additional 2 h. The mixture was then added to a pre-cooled mixture of 10% aqueous NaCl and DCM. The DCM layer was dried over sodium sulfate and concentrated to dryness to give an amber oil. The crude product was then purified on silica gel eluting with 5-50% DCM/methanol to give the pure product.

3-Benzyl-1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (6.00 g, 1 eq, 13.4 mmol) was dissolved in 20 mL DMF and then cooled to 0 C. To the mixture was then added sodium hydride (387 mg, 1.2 eq, 16.1 mmol) and stirring was continued at 0 C for an additional 30 min. To the reaction was then added 1-(bromomethyl)-2-nitrobenzene (2.90 g, 1 eq, 13.4 mmol) and stirring was continued at 0 C for an additional 1 h. The reaction was then added to a pre-cooled mixture of 10% NaCl and EtOAc. The EtOAc layer was separated, dried over sodium sulfate and concentrated to dryness. The crude product was purified on silica gel eluting with 0-50 hexanes/EtOAc to give the desired product.

3-Benzyl-1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-nitrobenzyl)oxy)-tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.50 g, 1 eq, 2.58 mmol) was dissolved in THF and cooled to 0 C. To the reaction was then added TBAF (674 mg, 1 eq, 2.58 mmol) at 0 C and stirred at 0 C for 1 h then warmed to room temperature over 2 h. The mixture was then poured into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was separated and dried over sodium sulfate. The crude product was purified on silica gel eluting with 5-25% DCM/methanol to give the desired product.

Benzyl (1-((2R,4S,5R)-5-(hydroxymethyl)-4-((2-nitrobenzyl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (35.0 mg, 1 eq, 70.5 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 min, a second aliquot of 10 uL was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise over 30 seconds to the reaction mixture at rxn t=35 minutes. Immediately, pre-weighed N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 eq, 345 μmol) was added in one portion as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation.

(Example 18)
Procedures for synthesizing Class VI peptide and non-peptide dGTP analogs.
Scheme for the synthesis of Class VI-dGTP constructs
Detailed Procedure for Non-peptide dGTP Analogs:

2-Amino-9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (0.50 g, 1 eq, 1.0 mmol) was dissolved in 5.0 mL of dry dimethylacetamide under argon. Oxirane (0.13 g, 3 eq, 3.0 mmol) was added at ambient temperature followed by sodium hydroxide (40 mg, 1 eq, 1.0 mmol) as a solid. The mixture was stirred at ambient temperature for 4 h. The mixture was diluted with 50 mL of EtOAc which was washed successively with 100 mL of water and 100 mL of brine. The EtOAc layer was dried over sodium sulfate and evaporated to leave a yellow oil. This was chromatographed on 40 g of silica using dichloromethane/methanol mixtures as eluent to give a white foam.

2-Amino-9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-1-(2-hydroxyethyl)-1,9-dihydro-6H-purin-6-one (200 mg, 1 eq, 370 μmol) was suspended in 5 mL of dry pyridine at ambient temperature under argon. Chloroformate (1 eq) was added as a solid. The mixture was heated to 95 C for 8 hours and cooled to ambient temperature. The solvent was removed in vacuo and the residue was diluted with 50 mL of EtOAc which was washed successively with 50 mL of water and 50 mL of brine. The EtOAc layer was dried over sodium sulfate and evaporated to leave a yellow oil. This was chromatographed on 40 g of silica using dichloromethane/methanol mixtures as eluent to give a white foam.

The alcohol starting material was dissolved in dry THF under argon at ambient temperature. Two equivalents of triethylamine were added. A solution of acyl chloride in THF was added dropwise at ambient temperature and the mixture was stirred for 18 hours. The solvent was removed in vacuo and the residue was diluted with 50 mL of EtOAc, which was washed successively with 50 mL of water and 50 mL of brine. The EtOAc layer was dried over Na2SO4 and evaporated to leave a light brown solid. This was chromatographed on a silica column using a dichloromethane/methanol mixture as eluent to give the corresponding ester.

The bis-silyl ether (1 eq.) was dissolved in dry THF under argon at ambient temperature. Triethylamine (8 eq.) was added rapidly followed by triethylammonium fluoride dihydrofluoride (6 eq.), also at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel was added and the mixture was evaporated on a rotary evaporator to a fine powder then loaded onto a silica column and eluted with a mixture of dichloromethane and methanol to give the nucleoside as a slightly yellow foam.

The nucleoside was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (1.5 eq.) was added. After 5 min, a second aliquot of 1.5 eq. was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (4 eq.) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 s. Immediately, a pre-weighed amount of N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (4 eq.) was added as a solid in one portion. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

Detailed procedure for peptide-dGTP analogs:

2-Amino-9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1.00 g, 1 eq, 2.02 mmol) was dissolved in 30 mL of dry N,N-dimethylacetamide under argon. 4-Bromobutanoic acid (337 mg, 1 eq, 2.02 mmol) was added at ambient temperature followed by sodium hydroxide (161 mg, 2 eq, 4.03 mmol) as a solid. The mixture was heated to 80 C and stirred for 12 h. The mixture was cooled to ambient temperature and diluted with 100 mL of EtOAc which was washed successively with 50 mL of water and 50 mL of brine. The EtOAc layer was dried over Na2SO4 and evaporated to leave a light brown solid which was chromatographed on a silica column using dichloromethane/methanol mixtures as eluent to give 4-(2-amino-9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-1-yl)butanoic acid as a white solid.

4-(2-amino-9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-1-yl)butanoic acid (1 eq) was suspended in 5 mL of dry pyridine at ambient temperature under argon. Chloroformate (1 eq) was added as a solid. The mixture was heated to 95 C for 8 hours and cooled to ambient temperature. The solvent was removed in vacuo and the residue was diluted with 50 mL of EtOAc which was washed successively with 50 mL of water and 50 mL of brine. The EtOAc layer was dried over sodium sulfate and evaporated to leave a yellow oil. This was chromatographed on 40 g of silica using dichloromethane/methanol mixtures as eluent to give a white foam.

The carboxylic acid was dissolved or suspended in dry THF at ambient temperature. To this solution was added 1.3 equivalents of triethylamine followed by 1.1 equivalents of diphenylphosphoryl azide. The mixture was heated to reflux for 20 hours and cooled to ambient temperature. Silica gel was added to the mixture and the solvent was evaporated to give a fine powder. This was loaded onto a column of silica gel and eluted with a mixture of EtOAc and dichloromethane to give the desired isocyanate as a colorless oil.

The bis-silyl ether (1 eq.) was dissolved in dry THF under argon at ambient temperature. Triethylamine (8 eq.) was added rapidly followed by triethylammonium fluoride dihydrofluoride (6 eq.), also at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel was added and the mixture was evaporated on a rotary evaporator to a fine powder then loaded onto a silica column and eluted with a mixture of dichloromethane and methanol to give the nucleoside as a slightly yellow foam.

The nucleoside was coevaporated with pyridine (1 mL x 3) and dried overnight under high vacuum. It was then dissolved in 1.5 mL trimethyl phosphate and 0.60 mL dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (1.5 eq.) was added. After 5 min, a second aliquot of 1.5 eq. was added. The mixture was stirred for an additional 30 min. A solution of tetrabutylammonium hydrogen diphosphate (4 eq.) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 s. Immediately, a pre-weighed amount of N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (4 eq.) was added as a solid in one portion. The mixture was stirred for 30 min after this addition and quenched with 8 mL of cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a small tube for FPLC separation, which was performed immediately after the EtOAc extraction. Final purification was by reverse phase HPLC.

Decaging of 3'-O-(2-nitro-benzyl)-dATP and homopolymer synthesis are shown in Figures 20 and 21. 25 uM 3'-O-(2-nitro-benzyl)-dATP (TriLink Technologies, San Diego, CA) was mixed with 1 uM oligonucleotide initiator (5'-biotin-TTTTTTGGCCTTTTUTAATAATAATAATAATTTTTT, IDT) along with 1x TdT reaction buffer (Thermo-Fisher), 2 U/uL terminal deoxynucleotidyl transferase (Thermo-Fisher) and 0.002 U/uL inorganic pyrophosphatase (Thermo-Fisher). Reaction volumes were subjected to 20-22 mW/cm2 of 365 nm light over various intervals and then placed at 37°C for 30 min. After quenching by the addition of 0.1 M EDTA, each time point was mixed with an equal volume of 2x Novex TBE-urea gel loading buffer (Thermo-Fisher), analyzed by polyacrylamide gel electrophoresis (15%), stained with Sybr Gold (Thermo-Fisher), and photographed under a UV transilluminator.

INCORPORATION BY REFERENCE References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS Various modifications of the invention, and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including references to the scientific and patent literature cited herein. The subject matter of this specification contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims (19)

1. A method for synthesizing a plurality of nucleic acid memory strands, comprising:
Providing an array of two or more nucleic acids linked to a substrate;
providing the array with blocked nucleotide analogs and a template-independent polymerase;
converting said blocked nucleotide analogs to unblocked nucleotide analogs by delivering addressable activation energy to a location on said array to remove a blocking group at the 3'-OH of said blocked nucleotide analogs, wherein said activation energy comprises light, reducing conditions, a pH change, or heat;
and extending one or more of said nucleic acids of said array with homopolymeric tracts of at least two repeating unblocked nucleotide analogues , wherein said blocking groups are 3'-O-blocking groups that cage the nucleotide analogues until removed to allow polymerization to proceed . The method of claim 1, wherein the blocked nucleotide analog comprises a removable blocking group on the 3'-OH of the deoxyribose or ribose of a nucleotide triphosphate and a non-removable modification on a purine or pyrimidine base of the nucleotide analog. The method of claim 1, wherein the multiple blocked nucleotide analogs are modified nucleotides of the same nucleobase that contain a removable 3'-O-blocking group and two or more non-removable molecular modifications that allow for differentiation between modified nucleotide analogs of the same nucleobase. stopping the elongation;
and extending the homopolymer tract with an additional homopolymer tract of two or more repeating nucleotides by delivering an addressable activation energy to the homopolymer tract in the presence of another plurality of blocked nucleotide analogs and the template-independent polymerase. 5. The method of claim 4 , wherein the elongation is stopped after a predetermined amount of time to obtain a desired length for the homopolymer tract. The method of claim 1, wherein the repeating nucleotides of the homopolymer tract are between 2 and 10. 5. The method of claim 4 , further comprising repeating the terminating and extending steps to synthesize a nucleic acid memory strand. 8. The method of claim 7 , wherein the nucleic acid memory strand is between 200 nucleotides in length and 5,000 nucleotides in length. 10. The method of claim 1, wherein a predetermined concentration of the blocked nucleotide analog is provided to obtain a desired length for the homopolymer tract. The method of claim 4 , wherein the homopolymer tract and the further homopolymer tract comprise different nucleobases. 8. The method of claim 7 , wherein the nucleic acid memory strand encodes a data set selected from the group consisting of a text file, an image file, and an audio file. The method of claim 11 , further comprising the step of displaying a human readable format of the data set on a monitor or using a printer. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is represented in base 2. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is represented in base 3. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is represented in base 4. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is represented in a base greater than base-4. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is indicated by the degree of decaging or the resulting tract length at individual steps of memory strand synthesis. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is read by DNA sequencing. 8. The method of claim 7 , wherein the data encoded in the nucleic acid memory strand is read by passing the nucleic acid memory strand through a nanopore.