JP7053009B2 - RNA aptamer screening method - Google Patents

Description

The present invention relates to RNA aptamer screening methods, kits, and libraries. More specifically, the present invention relates to a technique for significantly improving the screening efficiency of RNA aptamers by assembling and fixing clones of RNA aptamer candidates on the microsurface of a solid phase carrier.

Nucleic acid aptamers are single-stranded DNAs or RNAs that can specifically recognize targets, and are being actively researched for application to pharmaceutical development and diagnostic techniques. In particular, RNA is superior as a nucleic acid aptamer in that it can form a more complicated structure than DNA.

Examples of the method for acquiring a nucleic acid aptamer include the SELEX (Systematic Evolution of Ligands By EXPonential evolution) method. In the SELEX method, the target molecule is immobilized on a carrier and contacted with a population (library) of DNA or RNA having variations in the aptamer candidate sequence. Then, by recovering the carrier by centrifugation or the like, DNA or RNA that interacts with the target molecule immobilized on the carrier is obtained. In the SELEX method, the recovered nucleic acid is amplified by RT-PCR in the case of RNA and by PCR in the case of DNA, prepared again as a DNA library or RNA library, and subjected to the same sorting operation again. By repeating such a similar sorting operation, DNA or RNA (DNA aptamer or RNA aptamer) that specifically interacts with the target molecule is concentrated.

In the first library to be subjected to the SELEX method, a sequence containing a random sequence region of 30 bases or more is generally used as an aptamer candidate sequence. There are 4 ³⁰ variations of the random sequence of 30 bases, that is, 10 ¹⁸ or more, and the total amount of the aptamer candidate sequence containing all the sequences one molecule at a time is about 18 mg in terms of weight. Further, when a fixed sequence of about 40 bases required for binding a primer by PCR is added to the aptamer candidate sequence, the total amount becomes about 40 mg. Therefore, it is not practical to include all variations in the first library at the stage of the first sorting operation, considering the scale of the experiment, so usually only a small part of the huge number of combinations is the first. From there, the above-mentioned sorting operation is repeated to concentrate nucleic acids that specifically interact with the target molecule. For enriched aptamer candidate sequences, in order to further optimize the sequence that shows interaction with the target molecule, the sequences that are highly common among the enriched base sequences are fixed, while mutations are made to the surrounding sequences. The work of constructing the added library separately and repeating the sorting operation as described above is performed.

In the conventional SELEX method, it is necessary to chemically immobilize the target molecule on the carrier, but in recent years, various modification methods have been reported. For example, Patent Document 1 reports a method of performing selection by utilizing a change in molecular size accompanying the formation of a complex between a nucleic acid and a target molecule without fixing the target molecule. Further, Non-Patent Document 1 reports a method in which the side of the nucleic acid to be a library is immobilized on a carrier and selection is performed by utilizing a change in a fluorescent signal accompanying the binding between the nucleic acid and a target molecule. Emulsion PCR is used as a means for immobilizing the nucleic acid side on a carrier, and since it is possible to immobilize a large number of cloned DNAs of 100,000 copies or more on one carrier particle, it can be used with a target molecule. The efficiency of sorting is improved by increasing the change in the fluorescent signal associated with the binding.

International Publication No. 2003/10212

Angew.Chem.Int.Ed. (2014) 53,4796-4801.

Even if the selection of aptamer candidates from one library is repeated and narrowed down to a certain extent, usually, the work of newly arranging and constructing another library for further optimization and performing selection again is refrained. Therefore, in order to efficiently optimize aptamer candidates, one of the rate-determining steps is how to efficiently narrow down aptamer candidates from one library.

However, in the method of Patent Document 1, in order to narrow down the aptamer candidates from one library to a certain extent, the sorting work must be repeated many times, and the repeated sorting work is complicated and very inefficient. Further, although the method of Non-Patent Document 1 can improve the sorting efficiency, it can only sort DNA aptamers due to its design, and cannot be applied to the sorting of RNA aptamers.

Therefore, an object of the present invention is to provide a technique for further improving the screening efficiency of RNA aptamers.

The present inventor focused on the design of an RNA / DNA complex library in which RNA aptamer candidates transcribed using a DNA library immobilized on a fine particle carrier as a template are immobilized on the same fine particle carrier. As a result of diligent studies, a capture tag having a capture sequence and a linker was added to a library in which a large number of single-stranded DNA clones designed to have a capture sequence together with a template sequence of an RNA aptamer candidate were immobilized on a fine particle carrier. An extension reaction was carried out with the added primer to obtain a clone of double-stranded DNA containing the complementary sequence of the capture sequence on the fine particle carrier in the manner in which the capture tag was added, and then the double-stranded DNA was used as a template. By performing a transcription reaction to obtain an RNA aptamer to which a complementary sequence of the capture sequence is added as a transcript, and by capturing the transcript on a capture tag on the same fine particle carrier, each clone of the RNA aptamer candidate is captured. We succeeded in obtaining an RNA / DNA complex immobilized on the same microparticle carrier. Then, they have found that the screening efficiency of RNA aptamers is dramatically improved by using this RNA / DNA complex library. The present invention has been completed by further studies based on this finding.

That is, the present invention provides the inventions of the following aspects.
Item 1. RNA aptamer screening method including the following steps (A) to (C):
(A) An immobilized template double-stranded DNA library containing a plurality of tagged double-stranded DNA clones containing a template sequence of an RNA aptamer candidate according to the template sequence of the RNA aptamer candidate; the tagged. The template double-stranded DNA contains a complementary sequence of a promoter sequence of an enzyme having RNA polymerase activity, a capture sequence, and a template sequence of the RNA aptamer candidate from one end to the other, and a single-stranded DNA thereof. A double-stranded DNA composed of complementary strand DNA and a capture tag (Pt) containing a linker and the capture sequence attached to the one end of the complementary strand DNA, and a solid phase at the other end. Prepare an immobilized template double-stranded DNA library (LA) immobilized on a carrier; and immobilized on a specific microsurface of the solid phase carrier for each clone of the tagged template double-stranded DNA. And the process to do
(B) The template double-stranded DNA library (LA) is subjected to a transcription reaction to synthesize the RNA aptamer candidate to which the complementary sequence of the capture sequence is added, and the RNA aptamer candidate is attached to the capture tag (Pt). To prepare an immobilized RNA / DNA complex library (LB) by capturing it in
(C) The immobilized RNA / DNA complex library (LB) is brought into contact with the target substance, and the microsurface on which the clone of the RNA / DNA complex having the RNA aptamer candidate bound to the target substance is immobilized. The process of sorting.
Item 2. Item 2. The method according to Item 1, wherein the step (A) includes the following steps.
(A1) A DNA library containing a plurality of clones of the single-stranded DNA according to the template sequence of the candidate RNA aptamer, and each clone of the single-stranded DNA is placed on a specific microsurface of the solid phase carrier. A step of preparing an immobilized immobilized single-stranded DNA library (L0), and (A2) a capture tag (Pt) having the capture sequence and a linker for the immobilized single-stranded DNA library (L0). ) And a primer extension reaction using a primer (P) containing the promoter sequence to obtain the immobilized template double-stranded DNA library (LA).
Item 3. In the step (A1), the single-stranded DNA, its complementary strand DNA, or the double-stranded DNA composed of the single-stranded DNA and the complementary strand DNA is subjected to the presence of the solid phase carrier. Item 2. The method according to Item 2, wherein the immobilized single-stranded DNA library (L0) is obtained by subjecting to single molecule PCR using a primer.
Item 4. Item 2. The method according to Item 1, wherein the step (A) includes the following steps.
(A11) The capture sequence of the single-stranded DNA, its complementary strand DNA, or the double-stranded DNA composed of the single-stranded DNA and the complementary strand DNA in the presence of the solid phase carrier. And a step of subjecting to single molecule PCR using a primer (P) containing a capture tag (Pt) having a linker and the promoter sequence to obtain the immobilized template double-stranded DNA library (LA).
Item 5. A clone of an RNA / DNA complex having the RNA aptamer candidate bound to the target substance in the step (C), in which the solid phase carrier is a fine particle carrier and the fine surface is the surface of the fine particle carrier. Item 6. The method according to any one of Items 1 to 4, wherein the fine particle carrier on which the substance is immobilized is separated.
Item 6. Item 5. The method according to Item 5, wherein the sorting in the step (C) is performed by a cell sorter.
Item 7. An RNA having the RNA aptamer candidate bound to the target substance in the step (C), wherein the solid phase carrier is a substrate carrier, the microsurface is a specific microregion on the surface of the substrate carrier, and the microsurface is a specific microregion on the surface of the substrate carrier. The method according to any one of Items 1 to 4, wherein the microregion in which the clone of the / DNA complex is immobilized is specified.
Item 8. Items 1 to 1 in which the steps (A) to (C) are performed only once per one condition in the transfer reaction of the step (B) and one condition in contact with the target substance in the step (C). The method according to any one of 7.
Item 9. Item 6. The method according to any one of Items 1 to 8, wherein the promoter arrangement is a T7 promoter arrangement.
Item 10. Item 6. The method according to any one of Items 1 to 9, wherein the linker is a polyalkylene glycol chain.
Item 11. The single-stranded DNA containing the complementary sequence of the promoter sequence of the enzyme having RNA polymerase activity and the capture sequence, the complementary strand DNA thereof, or the double strand composed of the single-stranded DNA and the complementary strand DNA. Chain DNA and
A capture tag (Pt) having the capture sequence and a linker, a primer (P) containing the promoter sequence, and
A screening kit for RNA aptamers, including.
Item 12. A library containing a plurality of clones of tagged double-stranded DNA containing a template sequence of an RNA aptamer candidate according to the template sequence of the RNA aptamer candidate.
The tagged template double-stranded DNA comprises a complementary sequence of a promoter sequence of an enzyme having RNA polymerase activity, a capture sequence, and a template sequence of an RNA aptamer candidate from one end to the other. , A double-stranded DNA composed of the complementary strand DNA, and a capture tag (Pt) containing the linker and the capture sequence attached to the one end of the complementary strand DNA, and solidified at the other end. It is fixed to the phase carrier and
A library for screening RNA aptamers, which is immobilized on a specific microsurface of the solid phase carrier for each clone of the tagged template double-stranded DNA.
Item 13. The RNA aptamer candidate to which the complementary sequence of the capture sequence, transcribed from the tagged template double-stranded DNA, is complementarily bound to the capture tag (Pt) to form an RNA / DNA complex. Item 12. The RNA aptamer screening library according to Item 12.
Item 14. Item 6. The library for screening RNA aptamers according to Item 12 or 13, wherein the solid phase carrier is a fine particle carrier and the fine surface is the surface of the fine particle carrier.
Item 15. Item 12. The library for screening RNA aptamers according to Item 12 or 13, wherein the solid phase carrier is a substrate carrier and the microsurface is a specific microregion on the surface of the substrate carrier.

According to the present invention, the screening efficiency of RNA aptamer candidates can be dramatically improved by constructing a library of RNA / DNA complexes immobilized on the same fine particle carrier for each clone of RNA aptamer candidates. For example, since it is possible to narrow down several types from 10,000 or more types of RNA aptamer candidates in one screening, it is not necessary to repeat a large number of screenings under the same conditions in order to narrow down RNA aptamer candidates from one library.

An example of the screening method for the RNA aptamer of the present invention is schematically shown. An example of the tagged double-stranded DNA in the immobilized template double-stranded DNA library (LA) shown in FIG. 1 is schematically shown. An example of the step (A) for obtaining an immobilized template double-stranded DNA library (LA) is schematically shown. Another example of the step (A) for obtaining an immobilized template double-stranded DNA library (LA) is schematically shown. A schematic conceptual diagram of a nucleic acid portion in an example of step (A) is shown. A schematic conceptual diagram of the nucleic acid portion in the step (B) is shown. The schematic conceptual diagram of the screening kit of the RNA aptamer of this invention is shown. The sequences of the primers and DNA templates designed in Example 1 are shown. The result of the screening by Example 1 is shown. The sequences of the primers and DNA templates designed in Example 2 are shown. The result of the screening by Example 2 is shown. The sequence of RNA aptamer candidates narrowed down in Example 2 and the appearance rate (appearance frequency) of the sequence are shown. The change in the fluorescence signal due to the binding between the RNA aptamer candidate narrowed down in Example 2 and the target molecule is shown. The result of the screening by Example 3 is shown. Among the RNA aptamer candidates narrowed down in Example 3, the sequence not found in Example 2 and the change in the fluorescence signal due to the binding between the sequence and the target molecule are shown.

[1. RNA aptamer screening method]
The method for screening RNA aptamers of the present invention includes a step (A) for preparing an immobilized template double-stranded DNA library (LA), a step (B) for preparing an immobilized RNA / DNA complex library (LB), and a step (B). A step (C) of selecting RNA aptamer candidates is included. FIG. 1 schematically shows an example of the screening method for RNA aptamer of the present invention. As shown in FIG. 1, the immobilized template double-stranded DNA library (LA) prepared in step (A) is subjected to transcription in step (B) to immobilize RNA aptamer candidates. A clone of an RNA / DNA complex having an RNA aptamer candidate bound to the target substance is selected by being prepared into an RNA / DNA complex library (LB) and subjected to contact with the target substance in step (C). (In the example of FIG. 1, it is sorted).

In the immobilized template double-stranded DNA library (LA) prepared in the step (A), the tagged template double-stranded DNA shown as a schematic conceptual diagram in FIG. 2 is immobilized on the carrier. In the present invention, the step (A) includes an embodiment (FIG. 3) including the step (A1) and the step (A2), and an embodiment (FIG. 4) including the step (A11). Further, the details of the step (A) and the step (B) are shown in FIGS. 5 and 6, respectively, as schematic conceptual diagrams. Hereinafter, the method for screening the RNA aptamer of the present invention will be described in detail with reference to FIGS. 1 to 6.

In the present invention, "complementary" or "complementary" means having a base sequence capable of hybridizing to a nucleic acid having a certain base sequence under stringent conditions. The stringent conditions referred to here can be selected from known conditions and are not particularly limited, but for example, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum. Conditions such as albumin, 0.1% ficol (trade name), 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate coexist. Be done.

[1-1. Process (A)]
In step (A), an immobilized template double-stranded DNA library (LA) is prepared. As shown in FIG. 1, the immobilized template double-stranded DNA library (LA) contains numerous copies of clones of the tagged template double-stranded DNA immobilized on the microsurface of a solid phase carrier. In the illustrated embodiment, the solid phase carrier is a fine particle carrier. In this case, the microsurface of the solid phase carrier constitutes the entire surface of the microparticle carrier. As shown in FIG. 2, the tagged double-stranded DNA contains a complementary sequence of the promoter sequence of an enzyme having RNA polymerase activity, a capture sequence X', and a template for RNA aptamer candidates in the 3'-5'direction. It contains a single-stranded DNA containing sequences in this order, a double-stranded DNA composed of the complementary strand DNA thereof, and a capture tag (Pt) bound to one end of the complementary strand DNA. The solid phase carrier (not shown in FIG. 2) anchors the other end of the tagged template double-stranded DNA.

In the present invention, the RNA aptamer may be any molecule that can specifically bind to the target substance by having a high affinity, and the base sequence, the size of the molecule, and the three-dimensional structure are not particularly limited. Further, the base sequence may be an artificial sequence or a natural sequence. In the present invention, the candidates are arbitrarily set according to the use of the RNA aptamer (for example, nucleic acid medicine, diagnostic agent, etc.), and the template sequence of the set RNA aptamer candidate is designed.

In the immobilized template double chain DNA library (LA), the template sequences of RNA aptamer candidates are designed and compiled into a library with a plurality of sequences. In addition, as a plurality of sequences, a sequence designed by introducing a variation into a different similar sequence by introducing an arbitrary mutation into a specific location of a specific sequence can be mentioned. As a case of performing sequence design based on such variation expansion, for example, there is a case of optimizing an artificially designed RNA aptamer candidate. In addition, examples of the plurality of sequences include not only those expanded in variations into different similar sequences, but also sequences of RNA fragments obtained by randomly fragmenting long-chain RNA. Examples of sequence design based on such RNA fragments include the case of concentrating a sequence that specifically binds to a specific target substance (biomolecule such as protein) from a natural RNA sequence inherent in a cell. Be done.

The number of types of template sequences of RNA aptamer candidates set for one series of steps from step (A) to step (C) is not particularly limited, but for example, 3 million or less can be mentioned. From the viewpoint of obtaining a preferable screening efficiency, preferably 50,000 types or less, more preferably 30,000 types or less can be mentioned. Since the screening method for RNA aptamers of the present invention is extremely excellent in screening efficiency, the number of types of template sequences of RNA aptamer candidates includes 1000 or more, preferably 10,000 or more. Alternatively, as the number of types of template sequences of RNA aptamer candidates, for example, the number of combinations in which an arbitrary mutation is introduced into 1 to 10 bases (4 to 4 ¹⁰ patterns), preferably an arbitrary mutation is introduced into 2 to 9 bases. The number of combinations to be made (4 ² to ⁴⁹ patterns), more preferably the number of combinations into which any mutation is introduced into 3 to 8 bases (4 ³ to 4 ⁸ patterns), and even more preferably any mutation to 4 to 7 bases. The number of combinations ⁽ 44 to ⁴⁷ patterns) into which mutations are introduced can be mentioned.

The base length of the template sequence of the RNA aptamer candidate is not particularly limited, and is determined according to the base length of the target RNA aptamer. Specifically, the base length of the template sequence of the RNA aptamer candidate is, for example, 20 to 400 base length, preferably 30 to 100 base length.

In the immobilized template double-stranded DNA library (LA), a common sequence is designed as the complementary sequence and the capture sequence of the promoter sequence regardless of the template sequence of the RNA aptamer candidate. The promoter sequence may be any complementary sequence (antisense strand) of the double-stranded promoter region specifically recognized by RNA polymerase, and is designed according to the RNA polymerase used in step (B) described later. For example, T7 promoter sequence, T3 promoter sequence, K11 promoter sequence, SP6 promoter sequence and the like specifically recognized by RNA polymerase derived from Bacterophage can be mentioned. From the viewpoint of transfer stability and the like, a T7 promoter arrangement is preferably used.

The capture sequence X'is not particularly limited, and is appropriately designed so as not to cause undesired binding other than capturing RNA aptamer candidates in the step (B) described later. The base length of the capture sequence X'is not particularly limited, and may have a base length that can effectively capture RNA aptamer candidates in the step (B) described later. Specifically, the base length of the capture sequence X'includes 20 to 30 base lengths.

A tagged double-stranded DNA containing a single-stranded DNA having a complementary sequence of these promoter sequences, a capture sequence X', and a template sequence of an RNA aptamer candidate is a plurality of clones having a template sequence of one RNA aptamer candidate. Different surface of the microparticulate carrier for each clone, such that the copy binds to the surface of one microparticulate carrier and multiple copies of the clone with the template sequence of another RNA aptamer candidate bind to the surface of the other microparticulate carrier. It is fixed to. The number of copies of the clone bound to one fine particle carrier depends on the size of the fine particles, but for example, about one million copies can be mentioned.

In the illustrated embodiment, the case where the solid phase carrier is a fine particle carrier is exemplified, but the shape of the solid phase carrier is not limited to this. Examples of the solid phase carrier include a substrate carrier. When the solid phase carrier is a substrate carrier, the microsurfaces of the solid phase carrier constitute specific microregions on the surface of the substrate carrier. Then, a large number of copies of the clone of the tagged double-stranded DNA are aggregated and fixed in each of the partitioned microregions on the surface of the substrate carrier. That is, in the tagged template double-stranded DNA, multiple copies of a clone having a template sequence of one RNA aptamer candidate aggregate and bind to one microregion on a substrate carrier, and the template sequence of another RNA aptamer candidate is obtained. Each clone is immobilized on a different microregion on the same substrate carrier, such that multiple copies of the clones it has are aggregated and bound to other microregions on the same substrate carrier. Examples of the area per minute area include an area equivalent to a circle having a diameter of several tens of μm to several hundreds of μm.

The material of the solid phase carrier is not particularly limited, and examples thereof include glass, metal, polymer, and magnetic material. Further, the mode of fixing the DNA on the surface of the solid phase carrier is not particularly limited, and covalent bonds and non-covalent bonds are not limited. Further, it is preferable that the DNA strand is immobilized on the solid phase carrier via the adapter sequence.

[1-1-1. Process (A1)]
The step (A) for preparing the immobilized template double-stranded DNA library (LA) includes a step (A1) for preparing the immobilized single-stranded DNA library (L0) and an immobilized single-stranded DNA library (L0). It can be carried out by the step (A2) of subjecting to the primer extension reaction using the primer (P). FIG. 3 schematically shows an example of the step (A1) and the step (A2). Further, the details of the step (A2) are schematically shown in FIG.

The immobilized single-stranded DNA library (L0) does not yet have the complementary strand DNA in the above-mentioned immobilized template double-stranded DNA library (LA) and the capture tag (Pt) attached to it (see FIG. 2). A large number of copies of single-stranded DNA clones are immobilized on the microsurface of a solid-phase carrier. That is, the immobilized single-stranded DNA library (L0) contains the complementary sequence of the promoter sequence of the enzyme having RNA polymerase activity, the capture sequence X', and the template sequence of the RNA aptamer candidate in the 3'-5'direction. Multiple types of single-stranded DNA clones (see FIG. 5) containing RNA aptamer in this order are included according to the template sequence of RNA aptamer candidates, and each single-stranded DNA clone is on a specific microsurface of a solid phase carrier. It is fixed.

In the step (A1), the immobilized single-stranded DNA library (L0) may be prepared as a commercially available product, or may be prepared by preparing according to the step (A1) shown in FIG. According to the step (A1) shown in FIG. 3, the immobilized single-stranded DNA library (L0) is prepared by performing single molecule PCR of a template DNA fragment using a PCR primer in the presence of a carrier. Can be done. The template DNA fragment used in this case includes the above-mentioned single-stranded DNA (that is, the above-mentioned complementary sequence of the promoter sequence, the above-mentioned capture sequence X', and the above-mentioned RNA aptamer candidate template sequence in this order. It may be a double-stranded DNA), a complementary-stranded DNA (single-stranded) of the single-stranded DNA, or a double-stranded DNA composed of the single-stranded DNA and the complementary-stranded DNA. There may be.

For example, when the solid phase carrier is a fine particle carrier, emulsion PCR can be used as the single molecule PCR. In emulsion PCR, the above-mentioned template DNA fragment 1 molecule, a PCR primer (one of a forward primer and a reverse primer), and a PCR primer (the other of the forward primer and the reverse primer) are immobilized on the surface of the fine particle carrier 1. A large number of double-stranded DNAs are formed on the surface of the fine particle carrier by constructing an emulsion PCR system by adjusting the respective concentrations so that the individual particles are encapsulated in the water droplet emulsion in oil and performing the PCR reaction in the emulsion. Primer is fixed. DNA clones containing different RNA aptamer candidate template sequences for each emulsion partitioned as water droplets in oil are immobilized on the surface of the fine particle carrier. After the PCR reaction, the emulsion is destroyed, the fine particle carrier is recovered, and the double-stranded DNA is single-stranded to obtain an immobilized single-stranded DNA library (L0).

When the solid phase carrier is a substrate carrier, bridge PCR can be used as the single molecule PCR. In bridge PCR, the above-mentioned single-stranded DNA is amplified on the substrate. In this case, on the surface of the substrate on which the amplification reaction is performed, a set of two types of PCR primers that complementarily bind to the 5'end and 3'end of the single-stranded DNA is fixed to each of the partitioned microregions. Has been done. The above-mentioned single-stranded DNA bound to this PCR primer is PCRed on a substrate to form the same sequence, which is a complementary sequence of the promoter sequence of the enzyme having RNA polymerase activity, a capture sequence, and an RNA aptamer candidate. A large number of double-stranded DNA clusters containing the template sequence are formed in one small region on the substrate. By immobilizing the above-mentioned two types of PCR primers in a circular spot-shaped microregion having a diameter of several tens of μm to several hundreds of μm and appropriately adjusting the concentration of the above-mentioned single-stranded DNA, a plurality of types can be obtained in each of the partitioned microregions. Only one of the library sequences can be amplified. When the PCR primer is fixed in a small region, it is preferable to fix the PCR primer via a photocleavable group so that the immobilized nucleic acid species can be recovered later. The photocleavable group is not particularly limited as long as it is a linking group that can be cleaved by light irradiation, and examples thereof include a linking group having a 2-nitrobenzyl group as the photocleavable group. Such a photocleavable group is, for example, [4- (4,4'-Dimethoxytritylyloxy) butyramidomethyl] -1- (2-nitrophenyl) -ethyl] -2-cyanoethyl- (N, N-dissociotropicl) -phosphoramidite. It can be introduced using a reagent having a 2-nitro-4-substituted benzyl group of.

[1-1-2. Process (A2)]
As shown in the step (A2) of FIG. 3, in the step (A2), the above-mentioned immobilized single-stranded DNA library (L0) is subjected to a primer extension reaction using a primer (P) having a predetermined sequence design. To serve. As a result, the single-stranded DNA in the immobilized single-stranded DNA library (L0) becomes double-stranded DNA, and a single-stranded capture tag (Pt) is added to the end of the double-stranded DNA. An immobilized template double-stranded DNA library (LA) is prepared.

Specifically, as shown in FIG. 5, the primer (P) includes a capture tag (Pt) and a promoter sequence. The capture tag (Pt) has a capture sequence X'and a linker and is directly or indirectly bound to the promoter sequence via the linker. The promoter sequence is a sequence that binds to the complementary sequence of the promoter sequence of the single-stranded DNA in the immobilized single-stranded DNA library (L0). On the other hand, the capture tag (Pt) is a sequence non-complementary to the single-stranded DNA.

The capture sequence X'in the capture tag (Pt) is the single-stranded DNA of the above-mentioned immobilized single-stranded DNA library (L0) from the viewpoint of reliably capturing RNA aptamer candidates in the step (B) described later. It is preferably the same as the capture sequence X'. However, in the present invention, the capture sequence X'in the capture tag (Pt) is complementary to the transcription sequence of the capture sequence X'in the single-stranded DNA of the immobilized single-stranded DNA library (L0). As long as it is non-complementary to the single-stranded DNA, it does not have to be the same as the capture sequence X'in the single-stranded DNA of the immobilized single-stranded DNA library (L0). For example, the capture sequence X'in the capture tag (Pt) is one to a plurality (for example, 1 to 3) of the capture sequence X'in the single-stranded DNA of the immobilized single-stranded DNA library (L0). Base may be deleted, substituted, inserted, or added.

The linker in the capture tag (Pt) imparts swingability to the capture sequence X'by interposing in a hinge-like manner between the capture sequence X'and the promoter sequence, and RNA in the step (B) described later. Facilitates the capture of candidate aptamers. The linker is not particularly limited as long as it is non-complementary to the single-stranded DNA sequence, and examples thereof include polyalkylene glycol chains and oligonucleotide chains, and preferably polyalkylene glycol chains. The polyalkylene glycol chain has a structure represented by the general formula: —O— (RO) _n −, where n is an integer and represents the number of added moles of an alkylene oxide unit, and R represents an alkylene group. The alkylene group preferably has 2 or 3 carbon atoms, and more preferably a polyethylene glycol chain having 2 carbon atoms. Examples of the number of added moles n include 2 to 8, preferably 3 to 5, from the viewpoint of facilitating the capture of RNA aptamer candidates. The type of oligonucleotide chain is not limited to DNA and RNA. Preferred is polytimine. Examples of the base length of the oligonucleotide include, for example, 3 to 20 base lengths, preferably 5 to 10 base lengths, from the viewpoint of facilitating the capture of RNA aptamer candidates.

The promoter sequence is a sequence capable of forming a double-stranded promoter region specifically recognized by RNA polymerase together with a complementary sequence of the promoter sequence in the immobilized single-stranded DNA library (L0).

The primer (P) can be synthesized by a known method according to the above design. For example, it can be obtained by a method of chemical synthesis by a phosphoramidite method or the like, a method of digesting a nucleic acid containing a sequence to be contained in a primer (P) in advance with a restriction enzyme, or the like.

In the primer extension reaction, the primer (P) is complementarily bound to the single-stranded DNA, and the DNA strand is extended starting from the 3'end of this primer to synthesize the complementary strand DNA of the single-stranded DNA. Specifically, the immobilized single-stranded DNA library (L0) may be brought into contact with a buffer solution containing a primer (P), a DNA polymerase and a substrate deoxyribonucleotide, and the extension reaction may proceed under predetermined reaction conditions. .. The reaction conditions can be appropriately determined by those skilled in the art. For example, the primer (P) is complementarily bound to the single-stranded DNA under the conditions of 50 to 60 ° C. for 10 seconds to 30 seconds, and the optimum temperature of the DNA polymerase is obtained. Complementary strand DNA can be extended under the conditions of (for example, 68 to 72 ° C.) for 20 seconds to 1 minute. As a result, an immobilized template double-stranded DNA library (LA) in which a large number of clones of the tagged template double-stranded DNA having a capture tag (Pt) added on the solid-phase carrier is immobilized is obtained.

[1-1-3. Process (A11)]
The step (A) for preparing the immobilized template double-stranded DNA library (LA) can be performed by the above-mentioned steps (A1) and (A2), as well as by the following step (A11). FIG. 4 schematically shows an example of the step (A11).

The step (A11) can be performed in the same manner as the single molecule PCR performed in the above-mentioned step (A1), except that the primer (P) having a capture tag (Pt) is used instead of the PCR primer. That is, in the step (A11), the above-mentioned single-stranded DNA, its complementary strand DNA, or the double-stranded DNA composed of these single-stranded DNA and the complementary strand DNA is subjected to the presence of a solid phase carrier. It is used for single molecule PCR using the primer (P). As a result, PCR extension and PCR amplification as shown in FIG. 5 are performed, and an immobilized template double chain DNA library (LA) is obtained. Since the capture tag (Pt) is directly introduced into the library in single molecule PCR, an immobilized template double chain DNA library (LA) can be prepared in a simplified process.

[1-2. Process (B)]
In step (B), the above-mentioned immobilized template double-stranded DNA library (LA) is subjected to a transcription reaction to prepare an immobilized RNA / DNA complex library (LB). As shown in FIG. 1, in the immobilized RNA / DNA complex library (LB), RNA aptamer candidates were bound via a supplementary tag (Pt) in the above-mentioned immobilized template double-stranded DNA library (LA). Prepared in embodiments.

Specifically, as shown in FIG. 6, when the immobilized template double-stranded DNA library (LA) is subjected to a transcription reaction, RNA polymerase specifically binds to the promoter region of the tagged template double-stranded DNA. Tagged template RNA strands are synthesized using double-stranded DNA as a template to obtain RNA aptamer candidates.

Specifically, the immobilized template double-stranded DNA library (LA) may be brought into contact with a buffer solution containing RNA polymerase and a substrate ribonucleotide to allow the transcription reaction to proceed under predetermined reaction conditions.

The RNA polymerase is not particularly limited as long as it corresponds to the promoter region in the tagged double-stranded DNA. For example, RNA polymerase derived from bacteriophage can be mentioned, and more specifically, T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, and SP6 RNA polymerase can be mentioned. From the viewpoint of transcription stability and the like, T7 RNA polymerase is preferable. Furthermore, the RNA polymerase is not limited to the wild type, and any RNA polymerase having substrate specificity for the substrate ribonucleotide used in the present invention is genetically engineered to introduce mutations. May be.

The substrate ribonucleotide may be a natural product or a synthetic product. Examples of the synthesis of the substrate ribonucleotide include 2'-modified ribonucleotide (5'-phosphate ester of ribonucleoside having a modifying group at the 2'position of ribose), specifically, 2'-halogenation. One or more may be selected from the group consisting of ribonucleotides and 2'-alkoxyribonucleotides (for example, the carbon number of the 2'-alkoxy group is 1-3, preferably 1 and 2, more preferably 1). .. Those skilled in the art will appropriately select these synthetics, for example, those which are effective in the use of nucleic acid medicine for the purpose of improving chemical stability (biodegradation resistance), cell membrane permeability, molecular recognition, and the like. Can be done.

The nucleobase in the synthesis of the substrate ribonucleotide may be complementary to the template DNA. Specifically, one or more selected from adenine, guanine, uracil, and cytosine are used. The synthetic substrate ribonucleotide can be used, for example, by being appropriately mixed with a natural product of the substrate ribonucleotide.

Examples of the buffer solution include a solution containing an appropriate buffer component and a magnesium salt in water. Examples of the buffer component include phosphates such as trisacetic acid, trishydrochloric acid, sodium phosphate, and calcium phosphate. The final concentration of the buffer component is 5 mM to 100 mM, the pH is pH 6.0 to 9.5, and more preferably pH 7.0 to 8.0. Examples of the magnesium salt include magnesium chloride and magnesium acetate. Further, if necessary, a potassium salt such as potassium chloride and monopotassium glutamate, a surfactant such as DMSO, betaine, gelatin and Triton may be contained.

Even if the same template is used, different RNA aptamer candidates may be obtained depending on the solution conditions of the transcription reaction. Therefore, in addition to the above-mentioned components, the buffer solution is substantially subject to the solution conditions of the transcription reaction. Additives that have an adverse effect may be further contained. Examples of such additives include ethylene glycol, polyalkylene glycol, dextrin (Ficol), and biomolecules such as proteins having RNA binding properties, lipids, sugar chains, and other small molecule compounds. Of these additives, ethylene glycol has the effect of destabilizing the double-stranded structure, which is the standard structure of nucleic acid molecules, while stabilizing non-standard structures, including Hoogsteen base pairs. Is known, and it is considered that the binding property of the obtained RNA aptamer to the target substance may change with the stabilization and destabilization of such a structure. Therefore, ethylene glycol can be used to investigate the difference in the binding property of RNA aptamer candidate to the target substance depending on the presence or absence of addition to the transcription reaction solution.

The conditions of the transcription reaction are not particularly limited, and can be appropriately determined by those skilled in the art according to the optimum temperature of RNA polymerase, the scale of the library, and the like. For example, RNA chains can be synthesized under the conditions of 10 to 45 ° C., preferably 20 to 37 ° C., 10 minutes to 12 hours, preferably 10 minutes to 30 minutes.

Since the transcription reaction transcribes the capture sequence X'and the aptamer candidate template sequence in the tagged template double-stranded DNA, the RNA aptamer candidate, which is a transcript, has an embodiment in which the complementary sequence X of the supplementary sequence is added. Obtained at. Furthermore, in the synthesized RNA aptamer candidate molecule, the complementary sequence X of the capture sequence complementarily binds to the capture sequence X'of the adjacent supplementary tag (Pt) to form an RNA / DNA base pair (X / X). ') Is fixed by forming.

That is, the synthesized RNA aptamer candidate molecule is immobilized on the same surface as the surface of the microcarrier on which the tagged template double-stranded DNA presenting the template sequence is immobilized. This is because a large number of clones of the tagged double-stranded DNA are immobilized on the surface of the microcarrier, so that the RNA aptamer candidate molecule transcribed from the tagged double-stranded DNA is released into the transcription reaction system. This is due to the extremely high probability of being captured by a large number of capture tags (Pt) existing in the vicinity. The fact that the linker makes the capture sequence X'swayable in the capture tag (Pt) also contributes to the extremely high probability of capture of the RNA aptamer candidate molecule. Since the RNA aptamer candidate molecule transcribed on the surface of the same fine particle is rapidly fixed on the surface of the same fine particle, it is possible to suppress the exchange of RNA aptamer candidate molecule between different clones, that is, between the surfaces of different microcarriers. This prepares an immobilized RNA / DNA complex library (LB) in which a large number of clones of the RNA / DNA complex of the RNA aptamer candidate molecule are immobilized on a solid phase carrier.

That is, the immobilized RNA / DNA complex library (LB) is, as shown in FIG. 6, from one end to the other, a complementary sequence of the promoter sequence, a capture sequence X', and a template for RNA aptamer candidates. A capture tag (Pt) containing a linker sequence and a complementary sequence X', which contains a DNA strand containing a sequence and its complementary strand DNA and is bound to one end of the complementary strand DNA, and a capture tag (Pt). A clone of the RNA / DNA complex containing the candidate RNA aptamer complementarily bound to Pt) is immobilized on a solid phase carrier (not shown in FIG. 6, see FIG. 1) at the other end of the tagged template double-stranded DNA. Has been done. Then, a plurality of RNA / DNA complex clones are contained according to the sequence of the RNA aptamer candidate, and the RNA / DNA complex is immobilized on the individual surface of the fine particle carrier for each clone.

[1-3. Process (C)]
In step (C), the immobilized RNA / DNA complex library (LB) described above is brought into contact with the target substance. As shown in FIG. 1, among the clones of a plurality of types of RNA aptamer candidates included in the above-mentioned immobilized RNA / DNA complex library (LB), the target substance specifically binds to a specific clone. Further, the RNA aptamer candidate can be narrowed down by selecting the microsurface on which the clone of the RNA / DNA complex having the RNA aptamer candidate bound to the target substance is immobilized. In the selection of the fine surface, when the solid phase carrier is a fine particle carrier, the fine particle carrier on which the clone bound to the target substance is immobilized is separated, and when the solid phase carrier is a substrate carrier, the target substance is selected. Identify the microregions in which the bound clones are immobilized.

The target substance is not particularly limited, and the target substance is arbitrarily selected in the use of the RNA aptamer. For example, from a structural point of view, saccharides, lipids, oligopeptides, proteins, nucleic acids and the like can be mentioned. From a functional point of view, antigens, antibodies, ligands, receptors, biomarkers and the like can be mentioned.

As the conditions for contacting the immobilized RNA / DNA complex library (LB) with the target substance, conditions that do not impair the specific binding to be detected between the candidate RNA aptamer and the target substance can be appropriately selected. Specifically, the conditions can be selected in consideration of the pH of the test solution containing the target substance, the content of the solute and / or the coexisting component, and the like. As the conditions, it is preferable to select the same conditions (pH, content of solute and / or coexisting component, etc.) as in the actual application environment of RNA aptamer from the viewpoint of reliability of screening.

The target substance preferably has a signal group in order to facilitate detection. Examples of the signal group include a group derived from a fluorescent substance, a radioactive element-containing substance, a magnetic substance and the like. From the viewpoint of detectability and the like, the above-mentioned signal substance is preferably a fluorescent substance. Examples of the fluorescent substance include cyanine dyes such as fluorescein dyes and indocyanine dyes, fluorescent dyes such as rhodamine dyes; fluorescent proteins such as GFP; nanoparticles such as gold colloids and quantum dots. Examples of the above-mentioned radioactive element-containing substance include sugars, amino acids, nucleic acids and the like labeled with a radioisotope such as 18F. Examples of the above-mentioned magnetic substance include magnetic substances such as ferrichrome, ferrite nanoparticles, and nanomagnetic particles.

In the present invention, since a large number of clones of RNA aptamer candidates are immobilized on each of the small surfaces of the solid phase carrier, a large number of target substances can also be bound to the small surface. Therefore, when the target substance has a signal group, the signal change associated with the specific binding is large, so that the detection is easy and the selection efficiency is good.

When the solid phase carrier is a fine particle carrier, sorting using a cell sorter can be performed in the selection of RNA aptamer candidates specifically bound to the target substance. Specifically, a liquid containing an immobilized RNA / DNA complex library (LB) contacted with a target substance is flowed, passed through the focal point of laser light, and is specific to RNA aptamer candidates immobilized on individual fine particle carriers. By measuring the signal emitted by the specifically bound target substance, the specifically bound target substance can be quantitatively detected. Preferably, a preparative system based on the fluororescense activated cell sorting (FACS) method can be used. RNA aptamer candidates can be sorted by designating and sorting the fraction with the strongest signal intensity. By using the cell sorter, not only a specific fraction can be arbitrarily specified, but also the ratio of the fraction to the total RNA aptamer candidates used in the step (C) can be clarified. That is, it is possible to clarify how much screening efficiency was narrowed down in one step (C).

DNA or RNA sequence analysis can be performed on the separated fine particle carrier as it is or by freeing and collecting the fixed template DNA or RNA aptamer candidate. When the DNA is sequenced, the sequence of the DNA immobilized on the fine particle carrier can be directly analyzed, or the immobilized DNA can be amplified by PCR and then sequenced. When analyzing the sequence of RNA, stop the reverse transcription reaction using 2'-3'-dideoxynucleotide as a substrate together with RNA-degrading enzyme that distinguishes the type of nucleotide base and decomposes RNA, and after reverse transcription. Sequence analysis can be performed by utilizing amplification by PCR or the like. From the obtained sequence, a sequence that functions as an RNA aptamer can be selected.

When the solid phase carrier is a substrate carrier, in the selection of RNA aptamer candidates to which a target substance is specifically bound, a minute region can be specified using an imaging scanner, an array scanner, a microscope, or the like. Specifically, a substrate-like carrier containing an immobilized RNA / DNA complex library (LB) was brought into contact with a target substance, followed by a washing operation as necessary, and then immobilized on a specific microregion. It is possible to image the signal emitted by the target substance specifically bound to the RNA aptamer candidate. This makes it possible to identify a microregion in which a clone of an RNA / DNA complex having an RNA aptamer candidate bound to a target substance is immobilized. For the RNA / DNA complex in the specified microregion, if the RNA / DNA complex is previously immobilized on the substrate-like carrier via a photocleavable group, the identified microregion is irradiated with light. By cleaving the photocleavable group, the RNA / DNA complex can be released and recovered from the specified microregion.

According to the present invention, the screening efficiency of RNA aptamer candidates can be dramatically improved. Even when screening from the same library, the conditions for the transcription reaction in step (B) (eg, solution conditions, etc.) and the conditions for contact with the target substance in step (C) (eg, type of target substance, solution conditions, etc.). Although the RNA aptamer candidates to be narrowed down may change, the number of screenings is very small per one condition in the transcription reaction in step (B) and one condition in contact with the target substance in step (C) (((C). RNA aptamer candidates can be efficiently narrowed down from step A) to step (C) number of times). When the number of types of sequences in the library is, for example, 100,000 types or less, preferably 50,000 types or less, and more preferably 30,000 types or less, the number of screenings may be one. Therefore, it is not necessary to repeat a large number of screenings under the same conditions in order to narrow down RNA aptamer candidates from one library.

Further, according to the present invention, it is a functional aptamer in which two types of aptamers are combined, and the binding to the target molecule at the binding site of one aptamer affects the binding to the target molecule at the binding site of the other aptamer. To acquire a functional aptamer having an allosteric effect; to acquire a functional aptamer that changes the binding strength with a target molecule in response to the molecular environment in the solution such as ion concentration, pH, and the presence or absence of coexisting solute. Comprehensive analysis of the relationship between specific bases of RNA aptamer candidates and their contribution to recognition of target molecules; Creation of artificial RNA aptamers with unnatural base structures; Specific bases for interactions between proteins and metabolites and RNA Comprehensive analysis of the contribution of RNA bases; it is possible to obtain RNA sequences that interact with target molecules (proteins, etc.) from intracellular base sequences.

[2. RNA aptamer screening kit]
The RNA aptamer screening kit of the present invention is a kit for carrying out the above-mentioned RNA aptamer screening method. Therefore, the composition, usage, action, function, and other matters of the items included in or may be included in the RNA aptamer screening kit of the present invention are described in the above-mentioned "1. RNA aptamer screening method". It's a street.

As shown in FIG. 7, the screening kit for RNA aptamers of the present invention contains a primer (P) and a template DNA fragment as items. The template DNA fragment may be the above-mentioned single-stranded DNA (that is, single-stranded DNA containing a complementary sequence of the promoter sequence of an enzyme having RNA polymerase activity and a capture sequence X'). It may be a complementary strand DNA (single strand) of a single-stranded DNA, or a double-stranded DNA composed of the single-stranded DNA and the complementary strand DNA. The template DNA fragment can be customized according to the RNA aptamer candidate designed by the user, while the complementary sequence and the capture sequence of the promoter sequence are pre-designed to correspond to the sequence of the primer (P). The user designs a plurality of arbitrary RNA aptamer candidates, and prepares an appropriate solid-phase carrier and reagents necessary for the single-molecule PCR method, so that the DNA having the designed RNA aptamer candidate template sequence is single-stranded. An immobilized single-stranded DNA library (L0) bound to the 5'end of a DNA fragment can be constructed.

The immobilized single-stranded DNA library (L0) constructed by user customization uses a primer (P) and efficiently undergoes the steps described in the above-mentioned "1. RNA aptamer screening method" to efficiently perform RNA aptamers. Candidates can be narrowed down.

In the RNA aptamer screening kit of the present invention, any material, reagent, or the like for carrying out the method for screening the RNA aptamer of the present invention may be included as another item. Other items include, for example, solid phase carriers; primers, buffers, DNA polymerases, substrate deoxyribonucleotides for single molecule PCR; buffers, DNA polymerases, substrate deoxyribonucleotides for primer extension reactions; , Buffer solution, RNA polymerase, substrate ribonucleotide, ethylene glycol; target substance, signal reagent (for example, fluorescent reagent) for imparting a signal group (for example, fluorescent group) to the target substance and the like. These other items may be included in the kit alone or in combination of two or more.

Each item included in the screening kit for RNA aptamer of the present invention is provided in a liquid mode, a powder mode, a frozen mode, etc., in consideration of activity and / or storage stability, etc., according to their respective physical properties and the like. Further, each item may be packaged in a mode in which light-shielding, deaeration, sealing treatment and the like are appropriately performed in consideration of activity and / or storage stability, etc., according to each physical property and the like.

The RNA aptamer screening kit may further include protocol information indicating the content of the above RNA aptamer screening method. The information may be a printed matter on which the protocol is posted, or may be a printed matter on which information on the place of browsing or acquisition on the Web is posted so that the protocol can be browsed or obtained on the Internet or the like.

[3. RNA aptamer screening library]
The library for screening RNA aptamers of the present invention is a library useful for the above-mentioned method for screening RNA aptamers. Specifically, the immobilized template double-stranded DNA library (LA) used in the step (B) of the above-mentioned "1. RNA aptamer screening method" and the immobilized RNA used in the step (C). / DNA complex library (LB) can be mentioned. The composition, usage, action, function, and other matters of each Ibrali are as described in "1. RNA aptamer screening method" described above.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.

[overview]
Using the RNA aptamer (Spinach RNA) that binds to the fluorescent molecule 3'5'-difluoro-4-hydroxybenzylideneimidazolinone (DFHBI) and its derivative (DFHBI-1T), from crystal structure analysis in Spinach RNA. We constructed a library in which the bases around the DFHBI binding region that had been clarified were mutated, and attempted sequence optimization. The library was constructed by introducing a mutation into one base of the sequence and expanding the variation (Example 1) and by introducing a mutation into seven bases and expanding the variation (Example 2, Example 3). A template double-stranded DNA fragment containing variations in the sequence was prepared, immobilized on a microcarrier by emulsion PCR to prepare an immobilized single-stranded DNA library (L0) (step (A1)), and then became single-stranded. A double-stranded template DNA was synthesized from the DNA to prepare an immobilized template double-stranded DNA library (LA) (step (A)).

Then, a transcription solution containing DFHBI-1T is added to the immobilized template double-stranded DNA library (LA), and the Spinach RNA containing the mutation is immobilized on a microcarrier while undergoing a transcription reaction to immobilize the immobilized RNA / DNA complex. A library (LB) was obtained (step (B)). The transcription reaction was stopped, and the reaction solution containing the microcarriers was analyzed with a cell sorter, and the microcarriers showing the fluorescent signal of DFHBI-1T were sorted (sorted) and recovered (step (C)). When the number of recovered microcarriers was large, the microcarriers were purified again, and then the sequence of the template DNA immobilized on the microcarriers was directly analyzed by a next-generation sequence analyzer. When the number of recovered microcarriers was small, the recovered DNA was once amplified by PCR, and what kind of sequence was recovered was analyzed according to the conventional method of the next-generation sequence analyzer.

[Example 1: Screening and direct sequence analysis of aptamer candidates from single nucleotide polymorphism introducers to sites that interact with DFHBI-1T]
In the method for preparing the template double-stranded DNA fragment designed in this example and the step (step (A)) of preparing the immobilized template double-stranded DNA library (LA) from the immobilized single-stranded DNA library (L0). The sequence of the primer (P) used is shown in FIG. In FIG. 8, the base sequence region required for emulsion PCR is indicated by a broken line (the notation of the base is omitted), and the promoter sequence, the capture sequence X', and the specific base sequence of the Spinach RNA with the expanded variations (expressed in italics). ) Are described respectively. The template double-stranded DNA fragment was prepared by subjecting two DNAs having partially complementary sequences prepared by chemical synthesis to each other to extend the DNA (primer extension reaction). In the primer extension reaction, KOD-Plus-Ver. Of TOYOBO Co., Ltd. was used as an enzyme for extending DNA. 2 was used.

In this example, a template double-stranded DNA fragment in which one base that directly interacts with DFHBI is randomly mutated, which has been clarified from the crystal structure analysis of Spinach RNA, was designed. In this case, there are four types of sequence variations A, C, G, and T at the location represented by N in FIG. Based on the prepared template double-stranded DNA, the above-mentioned emulsion PCR operation was performed to prepare a microcarrier (immobilized single-stranded DNA library (L0)) on which the single-stranded DNA was immobilized (step (A1)). ). For emulsion PCR and subsequent purification and single chaining of microcarriers, Thermo Fisher Scientific's Ion OneTouch ^TM 2 System and Ion PGM ^TM Hi-Q ^TM View OT2 Kit were used. A DNA polymerase was added to the purified immobilized single-stranded DNA library (L0) together with a primer (P) to carry out a primer extension reaction to prepare an immobilized template double-stranded DNA library (LA) (step (A)). .. In the primer extension reaction in the step (A), 82.5 pmol of the primer (P) and 1 to 3 million immobilized single-stranded DNA libraries (L0) were mixed, and TOYOBO's KOD-Plus-Ver. No. 2 was used to perform an extension reaction at 56 ° C. for 30 seconds for only one cycle.

Then, the immobilized template double-stranded DNA library (LA) was purified by centrifugation, and the transcription reaction solution (40 mM Tris-hydrogenase (pH 8), 30 mM L-potassium glutamate, 8 mM magnesium chloride, 5 mM DTT, 2 mM spermidin, 0.01% Tween 20) was purified. ) And RNA polymerase were added to transcribe RNA, and a microcarrier (immobilized RNA / DNA complex library (LB)) on which the transcribed RNA was immobilized was prepared (step (B)). In step (B), T7 RNA polymerase manufactured by TAKARA is used, and a transcription reaction is carried out by adding a substrate ribonucleotide (40 μM) having a concentration lower than that of general transcription reaction conditions. 5 μM) was included. A transcription reaction was carried out at 25 ° C. for 10 minutes, and after adding competing DNA (double-stranded DNA having a T7 promoter sequence) to stop the transcription reaction, a fluorescent signal was transmitted by a cell sorter (BD FACSAria ^TM II manufactured by BD Biosciences). Analysis and fractionation of RNA / DNA complex library (LB) were performed (step (C)).

In step (C), when the fluorescence signal exhibited by the microcarrier was analyzed by a cell sorter with an excitation laser of 488 nm and a bandpass filter of 515-545 nm, multiple peaks with different fluorescence signal intensities were confirmed (Fig.). 9 (a1)). A peak region with the strongest fluorescence intensity (P3 region (abundance ratio of about 10%)) and a peak region showing fluorescence intensity of about 1/10 of the P3 region (P4 region (abundance ratio of about 30%)) are designated and respectively. About 300,000 microcarriers were separated. The obtained microcarriers are repurified using Biotin modified with a primer (P) and using Ion OneTouch ^TM ES of Thermo Fisher Scientific Co., Ltd., and together with the microcarriers before the preparative operation, the next generation. Direct sequence analysis was performed using a type DNA sequence analyzer (Ion PGM ^TM System: Thermo Fisher Scientific).

As a result of sequence analysis of about 10,000 to 25,000 sequences immobilized on the microcarriers fractionated from the P3 region and the P4 region, the microcarriers before the fractionation were found at the locations represented by N in FIG. The proportion of sequences having A, C, G, T (U) was A: C: G: T (U) = 36.57: 26.68: 16.14: 20.62 (FIG. 9 (FIG. 9). The ratio of the sequence of the microcarriers present in the P3 region to a2)) is A: C: G: T (U) = 0.10: 0.09: 99.63: 0.18 ( FIG. 9 (b2)) shows that the ratio of the sequence of the microcarriers present in the P4 region is A: C: G: T (U) = 96.62: 1.74: 0.12: 1. It was 52 (FIG. 9 (b1)). The sequence of the mutation site in the original Spinach RNA was G, and in the P3 region where the fluorescence signal intensity was strong, the same G sequence as the original Spinach RNA was concentrated to 99.63%. Further, in the P4 region showing a weak fluorescence signal, the sequence of A is concentrated to 96.62%, and it is possible to clearly select RNA aptamers based on the change in fluorescence signal intensity due to the difference in sequence. did it.

[Example 2: Screening and sequence analysis of aptamer candidates from sequence variations having a mutation of 7 bases]
In the method for preparing the template double-stranded DNA fragment designed in this example and the step (step (A)) of preparing the immobilized template double-stranded DNA library (LA) from the immobilized single-stranded DNA library (L0). The sequence of the primer (P) used is shown in FIG. In FIG. 10, the base sequence region required for emulsion PCR is indicated by a broken line (the notation of the base is omitted), and the promoter sequence, the capture sequence X', and the sequence of Spinach RNA with expanded variations (indicated in italics) are shown. .. The template double-stranded DNA fragment was prepared by performing a primer extension reaction in the same manner as in Example 1.

We designed a template double-stranded DNA fragment in which random mutations were introduced at 7 sites (7 bases) around the region where DFHBI binds in Spinach RNA. ^In this case, there are 47 = 16,384 types of sequence variations at the locations represented by N in FIG. In the same manner as in Example 1, a microcarrier (immobilized RNA / DNA complex library (LB)) on which the transcribed RNA was immobilized was prepared and analyzed by a cell sorter. As a result, although most of the carriers did not show a fluorescent signal (FIG. 11 (a)), a very small number of carriers, less than 0.01% of the total, specifically 45,861 carriers were analyzed. Three of the carriers showed the same strong fluorescence signal as confirmed in Example 1 (region shown in P5 of FIG. 11 (b)).

One million or more microcarriers were flushed through the cell sorter, of which 62 microcarriers contained in the P5 region were recovered. DNA was eluted by applying heat (95 ° C., 15 minutes) to the obtained microcarrier, amplified by PCR, and then sequence analysis was performed again using a next-generation DNA sequence analyzer. As a result, it was confirmed that eight kinds of sequences of SEQ ID NO: 1 (No. 1) to SEQ ID NO: 8 (No. 8) containing the same sequence as the original Spinach RNA have been concentrated (FIG. 12 (a). )). Of the 8 types of sequences, in the 7 types of sequences other than SEQ ID NO: 8, the sequences at 4 of the 7 sites into which the mutation was introduced were exactly the same sequence (single underlined sequence in FIG. 12 (b)). ). In addition, of the remaining three mutation sites, at two sites where mutations are added to the region where the original Spinach RNA forms a base pair, a GC base pair or an AT base pair should be formed. The sequence was preserved (double underlined sequence in FIG. 12 (b)).

Eight concentrated RNA aptamer candidates were transcribed in vitro to confirm binding to DFHBI-1T. Fluorescence signal (excitation wavelength) by mixing 20 nM DFHBI-1T and 4 μM RNA in a reaction solution (40 mM Tris hydrochloric acid (pH 8), 30 mM L-potassium glutamate, 8 mM magnesium chloride, 5 mM DTT, 2 mM spermidin, 0.01% Tween 20). As a result of measuring 470 nm (fluorescence wavelength 505 nm), an increase in the fluorescence signal associated with the binding between RNA and DFHBI-1T was confirmed in all seven sequences except SEQ ID NO: 8, which had no commonality in the sequences (s). FIG. 13). When the sequences of the immobilized template double-stranded DNA library (LA) before sorting (sorting) the microcarriers were analyzed and the appearance rates of each sequence before and after sorting were compared, fluorescence due to binding to DHFIBI-1T was compared. It was confirmed that the above 7 types of sequences showing an increased signal showed a concentration efficiency of 2,000 to 25,000 times (FIG. 12 (b)).

[Example 3: Screening of aptamer candidates that respond to the solution environment]
In the present invention, a transcription reaction is carried out on a template double-stranded DNA library (LA) under different solution conditions, or an immobilized RNA / DNA complex library (LB) is prepared by a normal transcription reaction, and then the microcarrier is centrifuged. By exchanging solutions while separating, aptamers can be selected based on the binding to target molecules under various solution conditions. Therefore, using the template double-stranded DNA library (LA) having 16,384 types of sequence variations used in Example 2, it was examined whether or not different aptamers could be selected depending on the solution environment. Specifically, the same as in Example 2 except that 30 wt% ethylene glycol was added to the transcription reaction solution when the template double chain DNA library (LA) was subjected to the transcription reaction (step (B)). Step (C) was performed.

In a solution environment in which a large amount of ethylene glycol is present, the double chain structure, which is the standard structure of nucleic acid molecules, tends to be destabilized, while the non-standard structure including Hoogsteen base pairs tends to be stabilized. It is known. Therefore, it is considered that the binding affinity for DFHBI-1T also changes depending on each sequence variation. When the immobilized RNA / DNA complex library (LB) after the transcription reaction was analyzed with a cell sorter in the same manner as in Example 2, most of the carriers showed no fluorescent signal (FIG. 14 (a)), but the whole. A very small number of carriers of less than 0.01%, specifically 4 of the 45,832 carriers analyzed, were present in the same P5 region as confirmed in Example 2 (. FIG. 14 (b). Of these, 75 microcarriers contained in the P5 region were recovered.

As a result of sequence analysis in the same manner as in Example 2, two types of sequences (SEQ ID NO: 9 (No. 9) and SEQ ID NO: 10 (No. 10)) were selected only under the solution condition to which 30 wt% ethylene glycol was added. )) Existed (FIG. 15 (a)). RNA was transcribed and purified in vitro in the same manner as in Example 2 for these two types of RNA, and the binding affinity with DFHBI-1T was evaluated in the presence or absence of 30 wt% ethylene glycol. Fluorescence signal (excitation wavelength) by mixing 20 nM DFHBI-1T and 4 μM RNA in a reaction solution (40 mM Tris-hydrochloric acid (pH 8), 30 mM L-potassium glutamate, 8 mM magnesium chloride, 5 mM DTT, 2 mM spermidin, 0.01% Tween 20). 470 nm, fluorescence wavelength 505 nm) was measured. As a result, it was confirmed that the fluorescence signal increased in the presence of 30 wt% ethylene glycol in both of the two sequences (FIG. 15 (b)). On the other hand, in the original Spinach RNA, the fluorescence signal decreased in the presence of 30 wt% ethylene glycol. That is, by using the method according to the present invention, aptamers that change the binding affinity with a target molecule in response to a specific solution environment are screened by comparing sequences selected under two conditions having different solution environments. It was shown to be possible.

Preferred embodiments of the present invention are as described above, but the present invention is not limited thereto, and various embodiments that do not deviate from the gist of the present invention are made elsewhere.

SEQ ID NOs: 1 to 10 are DNA sequences corresponding to the sequences of RNA aptamer candidates.

Claims (14)

RNA aptamer screening method including the following steps (A) to (C):
(A) An immobilized template double-stranded DNA library containing a plurality of tagged double-stranded DNA clones containing a template sequence of an RNA aptamer candidate according to the template sequence of the RNA aptamer candidate; the tagged. The template double-stranded DNA contains a complementary sequence of a promoter sequence of an enzyme having RNA polymerase activity, a capture sequence, and a template sequence of the RNA aptamer candidate from one end to the other, and a single-stranded DNA thereof. A double-stranded DNA composed of complementary strand DNA and a capture tag (Pt) containing a linker and the capture sequence attached to the one end of the complementary strand DNA, and a solid phase at the other end. Prepare an immobilized template double-stranded DNA library (LA) immobilized on a carrier; and immobilized on a specific microsurface of the solid phase carrier for each clone of the tagged template double-stranded DNA. And the process to do
(B) The template double-stranded DNA library (LA) is subjected to a transcription reaction to synthesize the RNA aptamer candidate to which the complementary sequence of the capture sequence is added, and the RNA aptamer candidate is attached to the capture tag (Pt). To prepare an immobilized RNA / DNA complex library (LB) by capturing it in
(C) The immobilized RNA / DNA complex library (LB) is brought into contact with the target substance, and the microsurface on which the clone of the RNA / DNA complex having the RNA aptamer candidate bound to the target substance is immobilized. The process of sorting. The method according to claim 1, wherein the step (A) includes the following steps:
(A1) A DNA library containing a plurality of clones of the single-stranded DNA according to the template sequence of the candidate RNA aptamer, and each clone of the single-stranded DNA is placed on a specific microsurface of the solid phase carrier. A step of preparing an immobilized immobilized single-stranded DNA library (L0), and (A2) a capture tag (Pt) having the capture sequence and a linker for the immobilized single-stranded DNA library (L0). ) And a primer extension reaction using a primer (P) containing the promoter sequence to obtain the immobilized template double-stranded DNA library (LA). In the step (A1), the single-stranded DNA, its complementary strand DNA, or the double-stranded DNA composed of the single-stranded DNA and the complementary strand DNA is subjected to the presence of the solid phase carrier. The method according to claim 2, wherein the immobilized single-stranded DNA library (L0) is obtained by subjecting it to single molecule PCR using a primer. The method according to claim 1, wherein the step (A) includes the following steps:
(A11) The capture sequence of the single-stranded DNA, its complementary strand DNA, or the double-stranded DNA composed of the single-stranded DNA and the complementary strand DNA in the presence of the solid phase carrier. And a step of subjecting to single molecule PCR using a primer (P) containing a capture tag (Pt) having a linker and the promoter sequence to obtain the immobilized template double-stranded DNA library (LA). A clone of an RNA / DNA complex having the RNA aptamer candidate bound to the target substance in the step (C), in which the solid phase carrier is a fine particle carrier and the fine surface is the surface of the fine particle carrier. The method according to any one of claims 1 to 4, wherein the fine particle carrier on which the substance is immobilized is separated. The method according to claim 5, wherein the sorting in the step (C) is performed by a cell sorter. An RNA having the RNA aptamer candidate bound to the target substance in the step (C), wherein the solid phase carrier is a substrate carrier, the microsurface is a specific microregion on the surface of the substrate carrier, and the microsurface is a specific microregion on the surface of the substrate carrier. / Identify the microregion where the clone of the DNA complex is immobilized,
The method according to any one of claims 1 to 4. Claim 1 in which the steps (A) to (C) are performed only once per one condition in the transfer reaction of the step (B) and one condition in contact with the target substance in the step (C). The method according to any one of 7 to 7. The method according to any one of claims 1 to 8, wherein the promoter arrangement is a T7 promoter arrangement. The method according to any one of claims 1 to 9, wherein the linker is a polyalkylene glycol chain. A library containing a plurality of clones of tagged double-stranded DNA containing a template sequence of an RNA aptamer candidate according to the template sequence of the RNA aptamer candidate.
The tagged template double-stranded DNA comprises a complementary sequence of a promoter sequence of an enzyme having RNA polymerase activity, a capture sequence, and a template sequence of an RNA aptamer candidate from one end to the other. , A double-stranded DNA composed of the complementary strand DNA, and a capture tag (Pt) containing a linker and the capture sequence attached to the one end of the complementary strand, and a solid phase at the other end. It is fixed to the carrier and
A library for screening RNA aptamers, which is immobilized on a specific microsurface of the solid phase carrier for each clone of the tagged template double-stranded DNA. The RNA aptamer candidate to which the complementary sequence of the capture sequence, transcribed from the tagged template double-stranded DNA, is complementarily bound to the capture tag (Pt) to form an RNA / DNA complex. The RNA aptamer screening library according to claim 11 . The library for screening RNA aptamers according to claim 11 or 12 , wherein the solid phase carrier is a fine particle carrier and the fine surface is the surface of the fine particle carrier. The library for screening RNA aptamers according to claim 11 or 12 , wherein the solid phase carrier is a substrate carrier and the microsurface is a specific microregion on the surface of the substrate carrier.

Images