JP7696809B2 - Nucleic acid aptamers and detection of influenza viruses - Google Patents

Description

The present disclosure relates to a nucleic acid aptamer for influenza virus type A/H1N1pdm09. The present disclosure relates to detection of influenza virus type A/H1N1pdm09 using the nucleic acid aptamer.

There are four types of influenza viruses: types A, B, C, and D, and types A and B cause seasonal influenza epidemics in humans every year. Type A influenza viruses are classified into 144 subtypes based on combinations of hemagglutinin (16 types, H1 to H16) and neuraminidase (9 types, N1 to N9), proteins that protrude from the surface of the virus, and are expressed as H1N1, H2N2, H3N2, etc.

In recent years, the influenza viruses causing seasonal influenza that infect humans and are prevalent are the influenza virus type A/H1N1pdm09 (hereinafter also referred to as A/H1N1pdm09), which became prevalent worldwide in 2009, and the influenza virus type H3N2 (hereinafter also referred to as H3N2). Although these influenza viruses do not show a high mortality rate, it has been reported that the clinical course differs for each subtype, that is, A/H1N1pdm09 is more likely to cause viral pneumonia and encephalopathy in children and young people than H3N2, while H3N2 is more likely to cause post-influenza bacterial pneumonia in relatively elderly people (Non-Patent Document 1). In addition, since multiple A subtypes may be prevalent at the same time, some patients may be infected with influenza virus twice in one season (Non-Patent Document 2).
Distinguishing between subtype A is highly important not only from the standpoint of treatment monitoring, but also from the standpoint of preventing repeated infection and superinfection.

To date, antibodies and aptamers have been developed to distinguish subtypes, and subtype discrimination test kits using these have also been developed (Patent Document 1, Non-Patent Document 3). However, because prevalent subtypes vary by year and region, and because HA and NA, which are the targets of subtype discrimination, are easily mutated because they exist on the surface of the virus, it is not always possible to distinguish prevalent strains.

JP 2012-100636 A

Influenza, Vol.19, No.2 (2018-11) P46-47 Influenza, Vol.21, No.1 (2020-3) P37 Acta Biomaterialia 9 (2013) pp8932-8941

Some existing aptamers have been shown to have reduced binding strength to prevalent influenza virus A subtypes, making it difficult to distinguish subtypes using these aptamers.
In order to create a diagnostic kit that can distinguish influenza virus A subtypes, it is necessary to keep up with the rapid mutations that occur in this virus, and so there was a need to develop an aptamer that reacts to the current circulating strain.

Although several aptamers have been developed to distinguish influenza A subtypes, no aptamers have been obtained that bind to the current epidemic A/H1N1pdm09 strain.

The present disclosure provides nucleic acid aptamers for influenza virus A/H1N1pdm09.
The present disclosure provides agents and methods for the detection of influenza A/H1N1pdm09 virus.

In one aspect, the present disclosure relates to a nucleic acid aptamer capable of binding to influenza virus A/H1N1pdm09, which consists of a nucleic acid containing a base sequence shown in any one of SEQ ID NOs: 4 to 11, or a base sequence in which one or several bases have been deleted, substituted or added in said base sequence.
In one aspect, the present disclosure relates to a nucleic acid aptamer capable of binding to influenza A/H1N1pdm09 virus, which consists of a nucleic acid comprising a base sequence that has been shortened so as to retain the structure of an end region portion that has binding ability to influenza A/H1N1pdm09 virus in a secondary structure constituted by a nucleic acid comprising a base sequence shown in any of SEQ ID NOs: 4 to 11, or a base sequence in which one or several bases have been deleted, substituted or added.

In one aspect, the present disclosure provides a nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, comprising:
The present invention relates to a nucleic acid aptamer, wherein the aptamer has a motif consisting of the 16th to 43rd bases from the 5' end of SEQ ID NO:4.
In one aspect, the present disclosure provides a nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, comprising:
The present invention relates to a nucleic acid aptamer having a first motif consisting of a base sequence represented by the 12th to 24th bases from the 5' end of SEQ ID NO: 6, and a second motif consisting of a base sequence represented by the 39th to 62nd bases from the 5' end of SEQ ID NO: 6.

In another aspect, the present disclosure relates to an agent for detecting influenza virus type A/H1N1pdm09, which comprises the nucleic acid aptamer of the present disclosure as an active ingredient.
In another aspect, the present disclosure relates to a diagnostic agent for influenza A/H1N1pdm09 virus, which contains the nucleic acid aptamer of the present disclosure as an active ingredient.
In another aspect, the present disclosure relates to a method for detecting influenza virus A/H1N1pdm09, comprising a step of allowing a nucleic acid aptamer of the present disclosure to act as an active ingredient on a test sample.
In another aspect, the present disclosure relates to a method for identifying at least one of an influenza virus subtype, strain, and clade, comprising a step of applying a nucleic acid aptamer of the present disclosure as an active ingredient to a test sample.

According to one aspect of the present disclosure, a nucleic acid aptamer capable of binding to influenza virus type A/H1N1pdm09 can be provided.
According to one aspect of the present disclosure, an agent and method for detecting influenza virus type A/H1N1pdm09 can be provided.

FIG. 1 is a graph showing the relationship between the number of cycles for selection and amplification of a target RNA aptamer and the amount of RNA bound to a virus. FIG. 2 shows the secondary structure (prediction) of the RNA aptamer. FIG. 3 shows the secondary structure (prediction) of an RNA aptamer. FIG. 4 is a graph showing the binding amount of the RNA aptamers of Experimental Examples 1 to 10 and Comparative Examples 1 and 2 to Ark19007 (H1N1pdm09), an A/H1N1pdm09 strain, and Ark1819002 (H3N2), an H3N2 strain. FIG. 5 shows the base sequences of the nucleic acids used in the examples. FIG. 6 shows the base sequences of the nucleic acids used in the examples. FIG. 7 shows the mutated portion of the mutant aptamer in the secondary structure (prediction) of the RNA aptamer (P30-10-h1-1). FIG. 8 is a graph showing the binding amount of the RNA aptamer (P30-10-h1-1) and its mutant aptamers to Ark19007 (H1N1pdm09), an A/H1N1pdm09 strain. FIG. 9 shows the mutated portion of the mutant aptamer in the secondary structure (prediction) of the RNA aptamer (P30-10-h1-1). FIG. 10 is a graph showing the binding amount of the RNA aptamer (P30-10-h1-1) and its mutant aptamers to Ark19007 (H1N1pdm09), an A/H1N1pdm09 strain. FIG. 11 shows the mutated portion of the mutant aptamer in the secondary structure (prediction) of the RNA aptamer (P30-10-h1-3). FIG. 12 is a graph showing the binding amount of the RNA aptamer (P30-10-h1-3) and its mutant aptamers to Ark19007 (H1N1pdm09), an A/H1N1pdm09 strain.

In the present disclosure, in one or more embodiments, the influenza A/H1N1pdm09 virus is a clinical isolate of influenza prevalent in the 2019/2020 season, and in one or more embodiments, is a clinical isolate of influenza A/H1N1pdm09 virus belonging to clade 6B.1A of the HA gene phylogenetic tree in the 2019/2020 season.

[Nucleic acid aptamer]
Nucleic acid aptamers are generally artificially engineered nucleic acid ligands that specifically bind to viruses, proteins, peptides, sugars, metal ions, small molecules, and the like.
The nucleic acid aptamer of the present disclosure has binding ability to influenza virus A/H1N1pdm09.
In the present disclosure, nucleic acid, in one or more embodiments, includes DNA, RNA, and analogs thereof.
In one or more embodiments, DNA and RNA may include chemically modified DNA and chemically modified RNA, respectively.
Even if the base sequence of the nucleic acid aptamer of the present disclosure is represented by DNA or RNA, it may be included in the nucleic acid aptamer of the present disclosure as a nucleic acid having a rearranged base sequence, for example, by converting thymine and uracil.

In one or more embodiments, the nucleic acid aptamer of the present disclosure consists of a nucleic acid containing a base sequence shown in any of SEQ ID NOs: 4 to 11, or a base sequence in which one or several bases have been deleted, substituted or added.
In one or more embodiments, the length of the nucleic acid aptamer of the present disclosure is preferably 100 bases or less, for example, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, or 40 bases or less. In one or more embodiments, the length of the nucleic acid aptamer of the present disclosure is 20 bases or more, 30 bases or more, 40 bases or more, or 50 bases or more. In one or more embodiments, the length of the nucleic acid aptamer of the present disclosure is 20 bases to 80 bases or 30 bases to 90 bases.
The base sequences of SEQ ID NOs: 4 to 11 were obtained by a method called the SELEX method, which is used to select a nucleic acid aptamer that binds strongly to a specific target substance. In the SELEX method, a nucleic acid library having random sequences is prepared, a nucleic acid that binds to the target substance is selected, and a cycle of PCR amplification is repeated multiple times to obtain a nucleic acid aptamer that binds strongly to the target substance. Currently, various improved SELEX methods have been reported.
The base sequences of SEQ ID NOs: 4 to 11 were obtained by performing a total of 10 cycles of selection by the SELEX method, and cloning the RNA pool that binds to a specific virus obtained. Specifically, a DNA library was synthesized with the central 30 bases of the sequence as a random region, PCR amplified, and then transcribed to create an RNA pool, and RNA that binds to the target influenza virus was selected and amplified from this RNA pool. By repeating this cycle 10 times, an aptamer with the base sequences shown in SEQ ID NOs: 4 to 11, which has high specificity and binding (affinity) to the A/H1N1pdm09 influenza virus, was obtained.

[Truncate aptamer]
In general, the secondary structure model of a nucleic acid aptamer can be predicted. The secondary structure of a nucleic acid aptamer is a stem-loop structure consisting of a tip loop structure and a stem structure. The site that actually binds to the target substance is thought to be mainly the stem region including the tip loop (hereinafter also referred to as the "tip region").
In one or a plurality of embodiments, secondary structure models of the nucleic acid aptamer of the present disclosure, which is composed of RNA having the base sequences of SEQ ID NOs: 4 to 11, are shown in FIGS.
In one or more embodiments, the nucleic acid aptamer of the present disclosure may include a nucleic acid aptamer in which the base sequences of SEQ ID NOs: 4 to 11 are shortened to the extent that the structure of the tip region of the secondary structure is not destroyed.
In one aspect, the present disclosure relates to a nucleic acid aptamer capable of binding to influenza A/H1N1pdm09 virus, which consists of a nucleic acid comprising a base sequence that has been shortened so as to retain the structure of a portion that has binding ability to influenza A/H1N1pdm09 virus in a secondary structure constituted by a nucleic acid comprising a base sequence shown in any of SEQ ID NOs: 4 to 11, or a base sequence in which one or several bases have been deleted, substituted or added.
In the present disclosure, in one or a plurality of embodiments, the portion capable of binding to influenza A/H1N1pdm09 virus is the tip region portion of the secondary structure described above.

In the nucleic acid aptamer of the present disclosure, the base sequence constituting the structure of the portion capable of binding to influenza virus A/H1N1pdm09, in one or a plurality of embodiments, can be any of the base sequences below.
A nucleotide sequence represented by the 15th to 44th bases from the 5' end of SEQ ID NO: 4 or 7;
A nucleotide sequence represented by the 11th to 48th bases from the 5' end of SEQ ID NO: 4 or 7 A nucleotide sequence represented by the 11th to 25th bases from the 5' end of SEQ ID NO: 6;
A nucleotide sequence represented by the 6th to 28th and 38th to 72nd bases from the 5' end of SEQ ID NO:6;
A base sequence represented by the 1st to 28th and 38th to 77th bases from the 5' end of SEQ ID NO:6.
In the nucleic acid aptamer of the present disclosure, the base sequence constituting the structure of the tip region capable of binding to influenza virus A/H1N1pdm09, in one or a plurality of embodiments, may be any of the base sequences below.
A nucleotide sequence represented by the 25th to 40th bases from the 5' end of SEQ ID NO: 4 or 7;
A nucleotide sequence represented by the 16th to 43rd bases from the 5' end of SEQ ID NO: 4 or 7;
A nucleotide sequence represented by the 15th to 44th bases from the 5' end of SEQ ID NO: 4 or 7;
A nucleotide sequence represented by the 11th to 48th bases from the 5' end of SEQ ID NO: 4 or 7;
A base sequence consisting of the 11th to 25th bases from the 5' end of SEQ ID NO:6.

In one or more embodiments, the "deletion, substitution, or addition" of one or several bases in the nucleic acid aptamer of the present disclosure is introduced to the extent that the structure of the portion in the secondary structure that has binding ability to influenza virus A/H1N1pdm09 is maintained, or is introduced at a location other than the structure of that portion.
In the present disclosure, several is, in one or more embodiments, two, three, four, or five.

In one or more embodiments, a nucleic acid aptamer having a deletion or substitution of a base in the base sequence of SEQ ID NO: 4 to 11 may have 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, or approximately the same or the same level of binding ability as a nucleic acid aptamer having a corresponding base sequence. In one or more embodiments, a nucleic acid aptamer having a deletion or substitution of a base in the base sequence of SEQ ID NO: 4 to 11 may have a higher level of binding ability than a nucleic acid aptamer having a corresponding base sequence, for example, a level of binding ability that is 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more higher.

In one or more embodiments, the binding ability of the nucleic acid aptamer of the present disclosure can be evaluated by the amount of binding to the A/H1N1pdm09 influenza virus relative to the amount of binding to the H3N2 (Ark1819002) influenza virus. In one or more embodiments, the amount of binding to the A/H1N1pdm09 influenza virus of the nucleic acid aptamer of the present disclosure relative to the amount of binding to the H3N2 (Ark1819002) influenza virus is 3 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more. In the present disclosure, the amount of binding to the influenza virus of the nucleic acid aptamer can be measured by the RT-qPCR method. Specifically, it can be measured by the method described in the Examples.

[Another embodiment of the truncated aptamer 1]
The present inventors have found that, in a nucleic acid aptamer formed of the base sequence shown in SEQ ID NO: 4, the base sequence shown at the 16th to 43rd bases from the 5' end of SEQ ID NO: 4 is involved in binding to influenza virus A/H1N1pdm09. Thus, in another aspect, the nucleic acid aptamer of the present disclosure is a nucleic acid aptamer having binding ability to influenza virus A/H1N1pdm09, and has a motif consisting of the base sequence shown at the 16th to 43rd bases from the 5' end of SEQ ID NO: 4.

In one or more embodiments, the motif of this aspect may have one or more loop structures, and preferably may have two loop structures and one stem structure located between the two loop structures. In one or more embodiments, the nucleic acid aptamer of this aspect has a structure formed by two loop structures and one stem structure located between the two loop structures.

In the present disclosure, the term "loop structure" refers to a single-stranded loop (circular) structure in a single-stranded nucleic acid that does not form base pairs. In one or more embodiments, the loop structure can also be said to be a loop structure that is located between two strands that form a stem structure and does not form base pairs. In one or more embodiments, the number of bases forming the loop structure is 5 to 50 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 35, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

In the present disclosure, the term "stem structure" refers to a chain-like structure formed by base-pairing one or more pairs of complementary bases in a single-stranded nucleic acid. In one or more embodiments, the stem structure may be a chain-like structure formed by completely or partially base-pairing some of the constituent bases or two or more consecutive bases. In one or more embodiments, the number of bases forming the stem structure is 2 or more and 20 or less (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
In one or a plurality of embodiments, the stem structure of the nucleic acid aptamer of this aspect contains two consecutive GC base pairs.

In one or more embodiments of the nucleic acid aptamer of this aspect, two or more bases may be added to each of the 5' end and the 3' end of the base sequence constituting the motif, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, 90, or 100 or more bases may be added. In one or more embodiments, some or all of the added bases may form base pairs to form a stem structure.

In one or more embodiments, the nucleic acid aptamer of this aspect includes the motif forming at least one loop structure, and a stem structure formed by bases added to each of the 5'-end and the 3'-end of the base sequence constituting the motif. In one or more embodiments, the number of bases forming the loop structure is 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
One or more non-limiting embodiments of the nucleic acid aptamer in this form include a nucleic acid comprising any one of the following base sequences:
A nucleotide sequence represented by the 15th to 44th bases from the 5' end of SEQ ID NO:4;
A nucleotide sequence represented by the 14th to 45th bases from the 5' end of SEQ ID NO:4;
A nucleotide sequence consisting of the 13th to 46th bases from the 5' end of SEQ ID NO:4;
A nucleotide sequence represented by the 12th to 47th bases from the 5' end of SEQ ID NO:4;
One or more particularly limited embodiments of the nucleic acid aptamer of this form include a nucleic acid having a motif consisting of the base sequence shown in the 16th to 43rd bases from the 5' end of SEQ ID NO: 4, and containing a base sequence having 85% or more identity with any of the above five sequences. In one or more embodiments, the aptamer retains the base sequence of the motif.

In one or more embodiments, the nucleic acid aptamer of this aspect has the motif described above and has 85% or more identity with the base sequence shown in the 1st to 58th bases from the 5' end of SEQ ID NO: 4. In one or more embodiments, but not particularly limited thereto, the nucleic acid aptamer of this aspect has one or more loop structures and one or more stem structures, and preferably has two loop structures formed by the motif, one stem structure located between the two loop structures, and stem structures located at the 5' end and 3' end of the motif.

In the nucleic acid aptamer of the present disclosure, a "nucleic acid aptamer having 85% or more identity to the base sequence shown in the SEQ ID NO" means that the nucleic acid aptamer is the specified base sequence itself, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the base sequence. The "% identity" of two nucleic acids can be determined by visual inspection or mathematical calculation. In one or more embodiments, the "% identity" of two nucleic acids can be performed using a readily available sequence comparison computer program. In one or more embodiments, computer programs include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), the BLAST package (Ausubel et al. (1999) ibid-Ch. 18), and FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410).
In one or more embodiments, the nucleic acid aptamer having 85% or more identity may retain the binding ability and/or the structure of the portion having the binding ability to the influenza virus A/H1N1pdm09. In one or more embodiments, the nucleic acid aptamer having 85% or more identity may have the above-mentioned binding ability of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, or approximately the same or the same level, as compared to the nucleic acid aptamer having the corresponding base sequence. In one or more embodiments, the nucleic acid aptamer having 85% or more identity may have the above-mentioned binding ability at a higher level than the nucleic acid aptamer having the corresponding base sequence, for example, the above-mentioned binding ability may be at a level higher than that of the nucleic acid aptamer having the corresponding base sequence, for example, at a level higher than that of the nucleic acid aptamer having 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[Another aspect 2 of the truncated aptamer]
The present inventors have found that, in a nucleic acid aptamer formed of the base sequence shown in SEQ ID NO: 6, the base sequence shown at 12th to 24th positions from the 5' end of SEQ ID NO: 6 and the base sequence shown at 39th to 62nd positions from the 5' end of SEQ ID NO: 6 are involved in binding to influenza A/H1N1pdm09 virus. Thus, in another aspect, the nucleic acid aptamer of the present disclosure is a nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, which has a first motif consisting of the base sequence shown at 12th to 24th positions from the 5' end of SEQ ID NO: 6 and a second motif consisting of the base sequence shown at 39th to 62nd positions from the 5' end of SEQ ID NO: 6.

In one or more embodiments, the first motif has a stem-loop structure. In one or more embodiments, the number of bases forming the loop structure is 5 or more and 10 or less (e.g., 5, 6, 7, 8, 9, or 10).

In one or more embodiments, the nucleic acid aptamer of this aspect may have a loop structure formed by a base sequence including the second motif. In one or more embodiments, the nucleic acid aptamer of this aspect may have 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) bases added to each of the 5' end and 3' end of the base sequence that forms the loop structure. In one or more embodiments, some or all of the added bases may form base pairs to form a stem structure.

In one or more embodiments, the nucleic acid aptamer of this aspect comprises a stem-loop structure formed by the first motif, a loop structure formed by a base sequence including the second motif, and a stem structure formed by bases added to each of the 5' end and 3' end of the base sequence constituting the loop structure, and the stem-loop structure formed by the first motif is connected to the loop structure formed by the base sequence including the second motif via the stem structure.
One or more non-limiting embodiments of the nucleic acid aptamer in this form include a nucleic acid comprising any one of the following base sequences:
A nucleotide sequence represented by the 6th to 28th and 38th to 72nd bases from the 5' end of SEQ ID NO:6;
A nucleotide sequence represented by the 5th to 28th and 38th to 73rd bases from the 5' end of SEQ ID NO:6;
Base sequences represented by the 4th to 28th and 38th to 74th bases from the 5' end of SEQ ID NO:6;
A nucleotide sequence represented by the 3rd to 28th and 38th to 75th bases from the 5' end of SEQ ID NO:6;
A nucleotide sequence represented by the 2nd to 28th and 38th to 76th bases from the 5' end of SEQ ID NO:6;
A base sequence represented by the 1st to 28th and 38th to 77th bases from the 5' end of SEQ ID NO:6.
One or more particularly non-limiting embodiments of the nucleic acid aptamer of this form include a nucleic acid having the above-mentioned first motif and second motif and containing a base sequence having 85% or more identity with any of the above-mentioned six sequences. In one or more embodiments, the aptamer retains the base sequences of the above-mentioned first and second motifs.

In one or more embodiments, the nucleic acid aptamer of this aspect has the above-mentioned first motif and second motif, and has 85% or more identity to the base sequence shown by the 1st to 77th bases from the 5' end of SEQ ID NO: 6. In one or more embodiments, but not particularly limited thereto, the nucleic acid aptamer of this aspect has one or more loop structures and one or more stem structures, and may preferably have a loop structure including the first motif, a stem-loop structure formed by the second motif linked to the loop structure, and stem structures located at the 5' end and 3' end of the loop structure including the first motif.

[Chemical modification]
Nucleic acid aptamers of the present disclosure may include chemically modified embodiments, as described above.
When the nucleic acid is RNA, the base sequence of the nucleic acid aptamer may contain chemically modified ribonucleotides in order to impart ribonuclease resistance. Such a nucleic acid aptamer can be obtained, for example, by substituting the 2'-OH group of the ribose moiety of the ribonucleotide in the nucleic acid aptamer with a fluoro group (2'-F) or a methoxy group (2'-OMe) by a conventional method, or by substituting the 2' position of the ribose moiety with hydrogen (2'-deoxy).
These chemical modifications are more effective on pyrimidine nucleotides since these sites are more susceptible to degradation by ribonucleases.
In one or more embodiments, these chemical modifications are introduced into a site in the secondary structure other than the structure of the portion having binding ability to influenza virus type A/H1N1pdm09. In one or more embodiments, these chemical modifications are made to the ribose group of the pyrimidine nucleotide in the loop region.

In one or more other embodiments, the chemical modification may be modification of the 5'-end and/or 3'-end of the nucleic acid aptamer with inverted deoxythymidine (idT) or polyethylene glycol (PEG), which improves the ribonuclease resistance of the aptamer RNA.
Since chemical modification can suppress the decrease in activity due to decomposition in the body, the nucleic acid aptamer of the present disclosure can also be made into a pharmaceutical composition that exerts an antiviral effect in the body.
In another aspect, the present disclosure relates to a pharmaceutical composition that contains the nucleic acid aptamer of the present disclosure as an active ingredient and exerts an antiviral effect against influenza virus A/H1N1pdm09.
In the present disclosure, in one or more embodiments, the phrase "containing the nucleic acid aptamer of the present disclosure as an active ingredient" may also include forms that contain the intermediates described below.

[Label]
In one or more embodiments, the nucleic acid aptamer of the present disclosure can be used to detect influenza virus A/H1N1pdm09, as described below.
The nucleic acid aptamers of the present disclosure include embodiments that are labeled for detection purposes.
The label can be appropriately selected depending on the detection method, and in one or more embodiments, examples of the label include fluorescent dyes, digoxigenin, digoxin, biotin, and radioactive substances.

[Intermediate]
In one aspect, the present disclosure relates to intermediates that can be converted in vivo or in vitro to the nucleic acid aptamers of the present disclosure.
The intermediate includes single-stranded DNA, double-stranded DNA, and RNA that contains the same base sequence as the nucleic acid aptamer of the present disclosure or a base sequence complementary to the base sequence and can be converted into the nucleic acid aptamer of the present disclosure by in vitro genetic engineering means or by a reaction in a living body or cell. These DNAs and RNAs may include chemically modified versions thereof and combinations thereof.
In one aspect, the present disclosure relates to a single-stranded DNA, double-stranded DNA, or RNA that contains a base sequence identical to or complementary to the nucleic acid aptamer of the present disclosure and that can be converted into the nucleic acid aptamer of the present disclosure.

[Manufacturing method]
In one or more embodiments, the aptamer of the present disclosure can be produced by chemical synthesis based on the base sequence. The DNA aptamer can be chemically synthesized from the terminal base using a DNA synthesizer and dNTP as a material. The synthesis of RNA requires protection of the 2' hydroxyl group of the ribose moiety, and various amidites have been developed as protecting groups, and the 2-cyanoethoxymethyl (CEM) group can be used.
In one or more embodiments, the aptamer of the present disclosure can be produced by a method (in vitro transcription method) in which the above-mentioned DNA corresponding to the RNA aptamer to be synthesized is chemically synthesized, this is amplified by PCR, and the RNA aptamer is synthesized from the amplified DNA by a transcription reaction using RNA polymerase.
In one or more embodiments, the aptamers of the present disclosure can be derived from complementary RNA by using an RNA-dependent RNA polymerase.

[Detection method]
In one or more embodiments, the nucleic acid aptamer of the present disclosure can be used to detect influenza virus A/H1N1pdm09, as described below.
In one aspect, the present disclosure relates to a method for detecting influenza virus A/H1N1pdm09 (hereinafter also referred to as the "detection method of the present disclosure"), which comprises a step of allowing a nucleic acid aptamer of the present disclosure to act on a test sample as an active ingredient. In one or more embodiments, the detection method of the present disclosure comprises contacting a test sample with a nucleic acid aptamer of the present disclosure, and measuring the binding between the nucleic acid aptamer of the present disclosure and influenza virus A/H1N1pdm09 in the sample. Alternatively, the detection method of the present disclosure comprises contacting a test sample with a nucleic acid aptamer of the present disclosure, and measuring whether the nucleic acid aptamer of the present disclosure binds to influenza virus A/H1N1pdm09 in the sample.
In one or more embodiments, acting as an active ingredient includes using it as an indicator for detection, binding to influenza A/H1N1pdm09 virus, or using it as a major factor in a detection mechanism.
In this disclosure, detection of a virus includes detecting the virus in a sample, and includes confirming the presence or absence of the virus in a sample suspected of containing the virus.
By confirming the presence or absence of the virus in a sample, at least one of the subtype, strain, and clade (lineage) of influenza virus contained in the sample can be identified or denied.
In one aspect, the present disclosure relates to a testing method for identifying or denying at least one of influenza virus subtypes, strains, and clades, comprising a step of acting a nucleic acid aptamer of the present disclosure on a test sample as an active ingredient. In one or more embodiments, the testing method of the present disclosure comprises contacting a test sample with a nucleic acid aptamer of the present disclosure and measuring binding between the nucleic acid aptamer of the present disclosure and the A/H1N1pdm09 influenza virus in the sample. Alternatively, the testing method of the present disclosure comprises contacting a test sample with a nucleic acid aptamer of the present disclosure and measuring whether the nucleic acid aptamer of the present disclosure binds to the A/H1N1pdm09 influenza virus in the sample.

In one or more embodiments, the test sample in the present disclosure may be a sample collected from a subject, such as a nasal cavity/pharyngeal swab or nasal secretion, or a solution obtained by infecting and growing such a sample in a chicken fertilized egg or cultured cell. In one or more embodiments, the test sample may be a sample that contains or may contain the A/H1N1pdm09 strain.

Detection Method Using Fluorescently Labeled Nucleic Acid Aptamer As one or more embodiments of the detection method of the present disclosure, a method using a fluorescently labeled nucleic acid aptamer of the present disclosure will be described.
The nucleic acid aptamer of the present disclosure labeled with a fluorescent dye such as fluorescein, rhodamine, or Texas Red is brought into contact with a test sample to carry out a binding reaction, and after removing unbound nucleic acid aptamer, the fluorescence or its intensity is detected and/or measured to detect or measure the amount of influenza A/H1N1pdm09 virus. For example, this can be carried out using a substrate on which either the labeled aptamer RNA or the test sample is immobilized.
Alternatively, a method in which neither the labeled aptamer nor the test substance is immobilized can be used to label either the aptamer of the present disclosure or the test sample with a substance that emits fluorescence upon binding, and detect or measure the fluorescence upon binding or its intensity. Examples of such fluorescent dyes include fluorescein, rhodamine, Texas Red, etc.

Detection Method Using Surface Plasmon Resonance (SPR) As one or more embodiments of the detection method of the present disclosure, a method using a nucleic acid aptamer of the present disclosure that is not fluorescently labeled will be described.
In the SPR method, minute changes in mass caused by binding between molecules immobilized on a sensor chip and molecules passing over the sensor chip can be detected as changes in the refractive index of reflected light. Either the aptamer RNA of the present disclosure or a target substance is immobilized on the gold thin film surface of a sensor chip by a known method, and a test substance or an aptamer RNA is supplied to the surface, thereby measuring the binding reaction of these molecules in real time.
An example of an apparatus that utilizes the SPR method is the Biacore T100 manufactured by GE Healthcare Biosciences.

Detection Method Using Immunochromatography As one or more embodiments of the detection method of the present disclosure, a method using immunochromatography will be described.
One embodiment is an immunochromatography method in which the nucleic acid aptamer of the present disclosure is used as a capture substance for immobilizing a virus at a detection site on a test strip (support) on which a sample is developed.
One embodiment is an immunochromatographic method in which a nucleic acid aptamer of the present disclosure is used as a capture substance to bind to and label viruses immobilized at a detection site on a test strip (support).
One embodiment is an immunochromatography method in which one or two types of nucleic acid aptamers of the present disclosure are used as a capture substance for fixing a virus at a detection site of a test strip (support) on which a sample is developed, and as a capture substance for binding to and labeling the virus fixed at the detection site of the test strip (support).
In one or a plurality of embodiments, the nucleic acid aptamer of the present disclosure used as a capture substance for labeling can be a nucleic acid aptamer of the present disclosure in a labeled form.

[Diagnostic Method]
The detection and testing methods of the present disclosure can be used in influenza diagnosis methods.
In another aspect, the present disclosure relates to a diagnostic method comprising confirming whether or not influenza A/H1N1pdm09 virus is present in a test sample using a detection method or testing method of the present disclosure, and determining, from the result, whether or not a subject who provided the sample is infected with the virus.

[Detection agents, diagnostic agents, kits]
In one aspect, the present disclosure relates to a detection agent, diagnostic agent, or kit containing the nucleic acid aptamer of the present disclosure as an active ingredient for use in the detection method, testing method, or diagnostic method of the present disclosure.
The detection agent can be used in the detection and testing methods of the present disclosure.
The diagnostic agent can be used in the diagnostic method of the present disclosure. One or more embodiments of the diagnostic agent include a companion diagnostic agent for a therapeutic agent against influenza A/H1N1pdm09 virus.
The kit can be used in the detection methods, testing methods, and diagnostic methods of the present disclosure.
When producing detection agents, diagnostic agents, and kits, they are used in combination with well-known pharma- ceutically acceptable diluents, stabilizers, other carriers, etc., as appropriate. These may include, in addition to the nucleic acid aptamer of the present disclosure, reagents and test strips used for detection.

The present disclosure may relate to one or more of the following non-limiting embodiments.
[1] A nucleic acid aptamer capable of binding to influenza virus A/H1N1pdm09, comprising a nucleic acid containing a base sequence shown in any one of SEQ ID NOs: 4 to 11, or a base sequence in which one or several bases have been deleted, substituted, or added in said base sequence.
[2] A nucleic acid aptamer capable of binding to influenza A/H1N1pdm09 virus, comprising a nucleic acid comprising a nucleotide sequence shown in any one of SEQ ID NOs: 4 to 11, or a nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequence, and which has been shortened so as to maintain the structure of a portion having binding ability to influenza A/H1N1pdm09 virus in a secondary structure constituted by the nucleic acid.
[3] The nucleic acid aptamer according to any one of [1], [2], and [12] to [16], wherein the nucleic acid is RNA.
[4] The nucleic acid aptamer according to any one of [1] to [3] and [12] to [16], wherein at least one site of the ribose moiety of the nucleotide constituting the aptamer is chemically modified.
[5] The nucleic acid aptamer according to [4], wherein the chemical modification is modification of the 2'-position of the ribose moiety with a fluoro group (2'-F) or a methoxy group (2'-OMe), or substitution with hydrogen (2'-deoxy).
[6] The nucleic acid aptamer according to any one of [1] to [5] and [12] to [16], wherein the 5'-end and/or the 3'-end is modified.
[7] A single-stranded DNA, double-stranded DNA, or RNA that contains the same base sequence as or is complementary to the nucleic acid aptamer described in any one of [1], [2], and [12] to [16], and can be converted into the nucleic acid aptamer described in any one of [1], [2], and [12] to [16].
[8] A detection agent for influenza A/H1N1pdm09 virus, comprising as an active ingredient the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16].
[9] A diagnostic agent for influenza A/H1N1pdm09 virus, comprising, as an active ingredient, the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16].
[10] A method for detecting influenza virus type A/H1N1pdm09, comprising a step of allowing a nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] to act as an active ingredient on a test sample.
[11] A testing method for identifying or denying at least one of an influenza virus subtype, strain, and clade, comprising a step of allowing a nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] to act as an active ingredient on a test sample.
[12] A nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, comprising:
The nucleic acid aptamer has a motif consisting of the base sequence represented by the 16th to 43rd bases from the 5' end of SEQ ID NO:4.
[13] The motif forms at least one loop structure,
The nucleic acid aptamer according to [12], further comprising a stem structure formed by bases added to each of the 5'-end and 3'-end of the base sequence constituting the motif.
[14] The nucleic acid aptamer according to [12] or [13], which has 85% or more identity with the base sequence represented by the 1st to 58th bases from the 5' end of SEQ ID NO: 4.
[15] A nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, comprising:
The aptamer has a first motif consisting of a base sequence represented by the 12th to 24th bases from the 5' end of SEQ ID NO:6, and a second motif consisting of a base sequence represented by the 39th to 62nd bases from the 5' end of SEQ ID NO:6.
[16] The nucleic acid aptamer according to [15], wherein the aptamer has a loop structure formed by a base sequence including the second motif, and further has a stem structure formed by bases added to each of the 5' end and the 3' end of the base sequence constituting the loop structure.
[17] The nucleic acid aptamer according to [15] or [16], which has 85% or more identity to the base sequence represented by the 1st to 77th bases from the 5' end of SEQ ID NO:6.

The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative and the present disclosure is not limited to these examples.

1. In vitro selection of aptamers specific to influenza virus A (H1N1) pdm09 (1-1) Acquisition and establishment of clinically isolated strains (1-1-1) Collection of specimens from influenza-infected individuals The nasal cavities of patients suspected of influenza infection in the 2018/2019 and 2019/2020 seasons were swabbed with a special swab or nasal secretions were collected, and the presence or absence of infection was determined using Arkray's influenza antigen detection kit, SPOTCHEM FLORA FluAB. If the patient's nasal secretion was positive for influenza A, the patient's nasal secretion was collected using a swab provided with the kit and suspended in 500 to 1000 μl of VTM (Copan) or Dulbecco's Modified Eagle Medium (Sigma-Aldrich) containing an antibiotic-antifungal drug mixture (Nacalai Tesque, hereafter referred to as antibiotic).
(1-1-2) Isolation and culture of influenza virus Madin-Darby Canine Kidney (MDCK) cells were seeded on a 12-well plate so that they would be 100% confluent the next day, and cultured overnight at 37°C in a 5% _CO2 environment. The next day, the supernatant of the 100% confluent cells was removed, and the cells were washed twice with 500 μl of PBS (-). 200 μl of diluted specimens collected from influenza-infected individuals were added to MDCK cells, and the cells were incubated for 30 minutes at 34°C in a 5% _CO2 environment to allow the virus to adhere to the cell surface. 800 μl of DMEM medium supplemented with antibiotics and 2.5 μg/ml acetyl trypsin (Sigma-Aldrich) was added, and the cells were cultured for 5 to 7 days at 34°C in a 5% _CO2 environment.
10 to 50 μl of the culture supernatant was sampled every few days of culture and measured using an influenza antigen detection kit (Arklay) to monitor the amount of virus in the culture supernatant and identify the type of influenza.
(1-1-3) Subtyping of clinical isolates, base sequence analysis of hemagglutinin gene The entire amount of culture supernatant of cells with increased viral load was collected, centrifuged at 3000 rpm, and the supernatant was collected (virus solution). Influenza virus RNA contained in 140 μl of the supernatant was extracted using a virus RNA extraction kit (Qiagen). Subtyping of the extracted RNA was performed by real-time PCR. For sequence analysis of the hemagglutinin gene, the virus RNA extracted with SuperScript (trade name) III One-Step RT-PCR System with Platinum Taq DNA Polymerase was reverse transcribed, and the full length of the hemagglutinin gene was amplified by PCR. It was confirmed that a fragment of the target size was amplified by agarose electrophoresis, and the base sequence of this fragment was analyzed. The primers and protocols used for real-time PCR, cloning, and sequencing were based on the Influenza Diagnostic Manual, 4th Edition.
The clinical isolates obtained were confirmed to be an H1N1pdm09 influenza virus strain belonging to clade 6B.1A of the HA gene phylogenetic tree (hereinafter also referred to as [Ark19007 (H1N1pdm09)]) and an H3N2 influenza virus strain belonging to clade 3C.2a (hereinafter also referred to as [Ark1819002 (H3N2)]).

The selection of nucleic acid aptamers by the SELEX method shown below in (1-2) to (1-4) was carried out with modified conditions based on the Influenza Diagnosis Manual, 4th Edition.
(1-2) Preparation of RNA Random Pool A single-stranded DNA (ssDNA) library with the central 30 bases as a random region was synthesized as shown below and used as a template (SEQ ID NO: 1). PCR was performed using a 5'-end primer (SEQ ID NO: 2) and a 3'-end primer (SEQ ID NO: 3).
SEQ ID NO: 1: AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG-(N)30-CCTTTCCTCTCTCTCCCTTCCTCTTC
SEQ ID NO: 2: AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG
SEQ ID NO: 3: AGAAGAGGAAGGAGAGAGAGGGAAAGG
Next, in vitro transcription was performed using a T7 Ampliscribe kit (Epicentre Technologies) to convert the amplified DNA library into an RNA library.

(1-3) In vitro Selection The RNA library (10 μg = 4 ³⁰ ≒ 1.15 × 10 ¹⁸ different RNA sequences) obtained in (1-2) above was dissolved in binding buffer (0.01 M HEPES, 0.15 M NaCl, pH 7.4). In order to promote equilibration of the RNA conformation, the RNA was denatured by treatment at 95°C for 2 minutes, and then cooled at room temperature for 10 minutes. In order to remove RNA that nonspecifically binds to the target, tRNA (total tRNA of E. coli (manufactured by Roche)) was added as a competitor to the RNA library solution, and then the target virus solution was added. In each selection cycle, negative selection was first performed using Ark1819002 (H3N2) as a counter virus. RNA that did not bind to this virus was collected, and then Ark19007 (H1N1pdm09) was reacted to perform positive selection. The molecular ratios of RNA (RNApool), viral protein (Counter/target) and tRNA (Competitor) used in each selection cycle are shown in Table 1.

100 μL of the mixture of RNA, viral protein, and tRNA was incubated at room temperature for 10 minutes, then passed through a wet nitrocellulose acetate filter (HA WP filter, 0.45 μm, diameter 13.0 mm, Millipore) attached to a "Pop-top" filter holder (Cytiva), and the RNA bound to the protein was captured on the filter. The filter was then washed with 1 ml of binding buffer. The RNA bound to the viral protein captured on the filter was eluted with elution buffer (0.01 M HEPES, 0.15 M NaCl, 7 M Urea, pH 7.4), and the RNA was purified by ethanol precipitation. The purified RNA was subjected to reverse transcription reaction in a 20 μl reaction solution using a primer (SEQ ID NO: 3), 0.4 mM dNTPs, 25 U PrimeScript (registered trademark) reverse transcriptase (TakaraBio), and the buffer provided with PrimeScript to obtain cDNA. The dNTPs and reverse transcriptase were added after denaturation and annealing steps (treatment at 95°C for 2 minutes, followed by incubation at room temperature for 5 minutes). Reverse transcription was performed at 42°C for 45 minutes.

80 μl of PCR mixture (PrimeSTAR® Max Premix (TaKaRaBio), 1 μM primer) was added to 20 μl of the mixture after reverse transcription reaction, and amplification by PCR was performed. The PCR reaction solution was heated at 95°C for 30 seconds, and then cycled at 95°C for 20 seconds; 54°C for 15 seconds; and 72°C for 15 seconds until a product band of the appropriate size was obtained (10-18 cycles). The PCR product obtained was purified by ethanol precipitation and used in the transcription reaction. In vitro transcription reaction was performed at 37°C overnight using a T7Ampliscribe kit. The transcribed RNA solution was treated with DNase I, and the reaction solution was fractionated on an 8% denaturing polyacrylamide gel. RNA was extracted from the gel, purified by ethanol precipitation, quantified, and used in the next selection and amplification cycle.

(1-4) Selection method and amplification cycles In order to obtain an RNA aptamer with specificity and high affinity for influenza virus, the amount of RNA and viral protein was changed for each selection cycle as shown in Table 1. In order to avoid the concentration of non-specifically binding RNA, a 96-well titer plate (Thermo Fisher) was used instead of a filter in each of the second, fourth, sixth, eighth, ninth and tenth selection cycles. For plate selection, first, 100 μg of the above viral protein per ml of borate buffer at pH 8.0 was immobilized in each well and blocked with BSA (1% stock solution). Then, each well was washed and used for selection.
The RNA pool obtained in the previous cycle was denatured in binding buffer at 95°C for 2 minutes. After cooling at room temperature for 10 minutes, tRNA was added and added to the wells to which the viral proteins were immobilized. After incubation for 10 minutes, the wells were washed with 300 μl of binding buffer (4 times in the second and third selection cycles, 6 times in the sixth and eighth selection cycles, and 8 times in the ninth and tenth selection cycles) to remove unbound RNA. Then, the viral protein-bound RNA was recovered with heated elution buffer (0.01 M HEPES, 0.15 M NaCl, 7 M Urea, pH 7.4), purified by precipitation with ethanol, and then regenerated by reverse transcription, PCR, and in vitro transcription.

(1-5) RNA Concentration Evaluation In order to evaluate the progress of the enrichment of high affinity aptamers and their specificity, the binding activity of the RNA pool in the 0th, 1st, 5th and 10th selection cycles was analyzed by filter binding quantification (RT-qPCR method). RNA pools for each selection cycle were prepared and bound to Ark19007 (H1N1pdm09) and Ark1819002 (H3N2) in solution. The binding reaction was performed by adding a 10-fold molar excess of E. coli tRNA as a non-specific competitive inhibitor, mixing 50 nM RNA and 1.58 μg of viral protein (500 nM in terms of hemagglutinin molecular weight). The reaction solution was passed through a nitrocellulose acetate filter, and the RNA bound to the virus was captured on the filter and washed with 2 ml of binding buffer. The viral protein-bound RNA captured on the filter was immersed in 200 μl of Elution buffer, heated and eluted, and the RNA was recovered by ethanol precipitation. The total amount of RNA was reverse transcribed in 20 μl of reaction solution containing 20 μM primer (SEQ ID NO: 3), 0.4 mM dNTPs, 25 U PrimeScript reverse transcriptase (TakaraBio), and PrimeScript buffer. 10 μl of real-time PCR reagent PowerSYBR Green Master Mix (Thermo Fisher), 1 μl of 5 μM forward primer, 1 μl of 5 μM reverse primer, and 9 μl of cDNA were mixed, and qPCR was performed using the reverse transcription product as a template. The binding activity was expressed as the ratio of the amount of RNA bound to the virus in the RNA pool in each selection cycle (Figure 1), with the amount of RNA bound to the virus in the 0th selection cycle set to 1. After the 10th selection cycle, the proportion of RNA captured by the filter was 7.0-fold higher for Ark19007 (H1N1pdm09) and 2.0-fold higher for Ark1819002 (H3N2).

(1-6) Analysis of Aptamers To obtain individual aptamers, the PCR products obtained in the 10th selection cycle were introduced into a TA cloning vector (Invitrogen) and transformed into Escherichia coli. Individual plasmid DNA was isolated using a plasmid purification kit (Promega), the DNA base sequence was decoded, and the RNA base sequence corresponding to the DNA base sequence was obtained. The RNA clones whose sequences were decoded were classified into a total of 10 types of sequences.

P30-10-h1-1: GGGAGAAUUCCGACCAGAAGUAGUAGCCCGGGGUGUGGGUUUAUGGUCGCCCCUUUCCUCUCUCUCCUCCUUCCUUCU (SEQ ID NO: 4)
P30-10-h1-2: GGGAGAAUUCCGACCAGAAGGCGCGAUUGUGGUUGUGGUGGGUGGGCGCGCCUUUCCUCUCUCCUUCCUUCU (SEQ ID NO: 5)
P30-10-h1-3: GGGAGAAUUCCGACCAGAAGUGUCGAUGUGUAUCUUAUUUGUUUGUUUGUUUGUUUGUUUGUCCUUUCCUCUCUCUCCUUCCUCUCUUCU (SEQ ID NO: 6)
P30-10-h1-4: GGGAGAAUUCCGACCAGAAGGCUAUGGGUUGAGUGUCUGUAUGGGUGGGUGCCUUUCCUCUCUCCUUCCUCCUUCCUUCU (SEQ ID NO: 7)
P30-10-h1-5: GGGAGAAUUCCGACCAGAAGUCCCCUCCCUCGUAUCGUAUGUGCGUUUGCCCUUUCCUCUCUUCCUUCCUCUUCU (SEQ ID NO: 8)
P30-10-h1-6: GGGAGAAUUCCGACCAGAAGUAGUAGCCCGGGGUGUGGGUUUAUGGCCGCCCCUUUCCUCUCUCUCCUUCCUUCU (SEQ ID NO: 9)
P30-10-h1-7: GGGAGAAUUCCGACCAGAAGUAGUAGCCCGGGGUGUGGGUUUAUGGUCGUCCCUUUCCUCUCUCCUUCCUCUC (SEQ ID NO: 10)
P30-10-h1-8: GGGAGAAUUCCGACCAGAAGGCGCGAUUGUGUUGUGGUGGGUGGGCGCGCCUUUCCUCUCUCCUUCCUUCU (SEQ ID NO: 11)
P30-10-h1-9: GGGAGAAUUCCGACCAGAAGGAACA UUUGGGGUGGGUGGGUGGGUGGCUGUUCCUUUCCUCUCUCUCCUUCCUUCU (SEQ ID NO: 12)
P30-10-h1-10: GGGAGAAUUCCGACCAGAAGGGUCGGUGUAUAAUUGUAGUUUUGUUGUUGUUGUUGUUGUUGCCUUUCCUCUCUCCUUCCUCUCUCU (SEQ ID NO: 13)

Among the RNA base sequences selected targeting Ark19007 (H1N1pdm09), aptamer P30-10-h1-1 (SEQ ID NO: 4) accounted for 63.5% of the total, and aptamer P30-10-h1-2 (SEQ ID NO: 5) accounted for 17.3% of the total. P30-10-h1-3 (SEQ ID NO: 6) and P30-10-h1-4 (SEQ ID NO: 7) each accounted for 3.8% of the total, and P30-10-h1-5 to P30-10-h1-10 (SEQ ID NO: 8 to 13) each accounted for 1.9% of the total. Furthermore, P30-10-h1-6 and P30-10-h1-7 had sequences that differed from P30-10-h1-1 by only one base. The secondary structure of the obtained aptamers was predicted using the secondary structure analysis software "The mfold Web Server" (analysis can be performed at http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form). Although multiple secondary structures may be predicted for each aptamer RNA, the most stable predicted structure for each aptamer RNA is shown (Figures 2 and 3).

2. Analysis of affinity between aptamers and each viral protein by filter binding assay (RT-qPCR) The binding efficiency of the 10 aptamers selected in 1 above to influenza virus clinical isolate Ark19007 (H1N1pdm09) was determined by filter binding assay (1-5 above) (Comparative Examples 1 and 2, and Experimental Examples 1 to 10). The results are shown in Table 2 and Figure 4.
As comparative examples, D-12 (SEQ ID NO: 14) and D-26 (SEQ ID NO: 15), which are known aptamers specific to the hemagglutinin of the A/California/07/2009 (H1N1) pdm09 influenza virus, were used (JP Patent Publication No. 2012-100636). Note that D-26 was used as D26 (2'-F) in which the OH at the 2' position of the pyrimidine base was replaced with F.
Aptamer D12: GGAGCUCAGCCUUCACUGCCAAAGUGCGAGGCAGUGUGGUGUGCUGUCCUACGAGUUCUAAAGUUCGUUAGGAAGGCAGCUCAACAUGUUUAACAGGCACCACCGUCGGAUCC (SEQ ID NO: 14)
Aptamer D26: GGAGCUCAGCCUUCACUGCCAAAAGUUAGGCCAGCAAAUUGCGAGCUGAUCCGGUGACUGGCUACAGGAGGCCUUGUCCACGGCCGUAUUGGCACCACCGUCGGAUCC (SEQ ID NO: 15)

Aptamers P30-10-h1-1, P30-10-h1-2, P30-10-h1-3, P30-10-h1-4, P30-10-h1-5, P30-10-h1-6, P30-10-h1-7, and P30-10-h1-8 (Experimental Examples 1 to 8) showed higher binding to Ark19007 (H1N1pdm09) than to Ark1819002 (H3N2). P30-10-h1-1, P30-10-h1-2, P30-10-h1-3, P30-10-h1-4, P30-10-h1-5, P30-10-h1-6, P30-10-h1-7 and P30-10-h1-8 were shown to be aptamers with stronger binding strength than the existing aptamers D12 and D26 (2'-F). The amount of aptamer RNA binding to Ark19007 (H1N1pdm09) and Ark1819002 (H3N2) was as shown in Table 2.

<Binding evaluation of mutant aptamers>
In order to identify the sequence required for the binding of the aptamer to the target substance, mutant aptamers were prepared by deleting, inserting or mutating a portion of the sequence as shown in Figure 6, and their binding to Ark19007 (H1N1pdm09) was evaluated.
Specifically, aptamers obtained by the SELEX method (P30-10-h1-1 (sequence number 4) and P30-10-h1-3 (sequence number 6)) were modified with biotin, and the binding ability of the mutant aptamer to viral proteins was evaluated by measuring the extent to which the binding of the biotin-modified aptamer to viral proteins was inhibited by the addition of unlabeled mutant aptamer.

Based on the predicted secondary structure of the RNA aptamer as shown in FIG. 2, the region (motif) required for binding to the viral protein is predicted to be a stem-loop structure sequence.
First, in P30-10-h1-1, four mutant aptamers (SEQ ID NOs: 16 to 19) were prepared by deleting the regions shown in FIG. 7, and the aptamer binding inhibitory activity of these was evaluated.
Specifically, P30-10-h1-1_D1 (SEQ ID NO: 16) has 16 bases deleted from the 3' end of P30-10-h1-1 (SEQ ID NO: 4). P30-10-h1-1_D2 (SEQ ID NO: 17) has the 7th to 10th bases and the 49th to 52nd bases deleted from the 5' end of P30-10-h1-1 (SEQ ID NO: 4). P30-10-h1-1_D3 (SEQ ID NO: 18) has the 18th to 24th bases deleted from the 5' end of P30-10-h1-1 (SEQ ID NO: 4). P30-10-h1-1_D4 (SEQ ID NO: 19) has the 30th to 35th bases deleted from the 5' end of P30-10-h1-1 (SEQ ID NO: 4).
As a result, the mutant aptamers P30-10-h1-1_D3 (SEQ ID NO: 18) and P30-10-h1-1_D4 (SEQ ID NO: 19) did not exhibit binding inhibitory activity against the biotin-modified aptamer. Therefore, it was presumed that the sites deleted in these mutant aptamers are regions (motifs) necessary for binding to viral proteins (FIG. 8). In other words, the base sequences shown at 17th to 24th bases from the 5' end and 30th to 35th bases from the 5' end of SEQ ID NO: 4 are predicted to be core sequences (motifs) that may be necessary for binding to viral proteins.

Since these refer to two loop structures, six mutant aptamers were prepared by adding mutations to the bases in the region connecting the two loop structures, as shown in Figure 9 (SEQ ID NOs: 20 to 25). Of the mutant aptamers prepared, del1 and del2 are deletion type (the bases in the square frame in Figure 9 are deleted), in1 and in2 are insertion type (insertion at the arrow in Figure 9), and mt1 and mt2 are point mutation type (the bases in the square frame in Figure 9 are replaced). Specifically, P30-10-h1-1_del1 (SEQ ID NO: 20) was prepared by deleting the 27th base (C) and the 38th base (G) from the 5' end of P30-10-h1-1 (SEQ ID NO: 4). P30-10-h1-1_del2 (SEQ ID NO: 21) is obtained by deleting the 25th and 26th bases (AG) and deleting the 38th and 39th bases (GU) from the 5'-terminus of P30-10-h1-1 (SEQ ID NO: 4). P30-10-h1-1_in1 (SEQ ID NO: 22) is obtained by inserting one base (A) between the 27th base (C) and the 28th base (C) from the 5'-terminus of P30-10-h1-1 (SEQ ID NO: 4) and inserting one base (A) between the 37th base (G) and the 38th base (G). P30-10-h1-1_in2 (SEQ ID NO: 23) has two bases (CC) inserted between the 27th base (C) and the 28th base (C) from the 5' end of P30-10-h1-1 (SEQ ID NO: 4), and two bases (GG) inserted between the 37th base (G) and the 38th base (G) from the 5' end of P30-10-h1-1_mt1 (SEQ ID NO: 24) has the 39th base (U) from the 5' end of P30-10-h1-1 (SEQ ID NO: 4) mutated to C. P30-10-h1-1_mt2 (SEQ ID NO: 25) has the 27th base (C) from the 5' end of P30-10-h1-1 (SEQ ID NO: 4) mutated to A.
The binding inhibitory activity of these mutant aptamers against H1N1pdm09 viral proteins was evaluated, and none of the mutant aptamers exhibited binding inhibitory activity ( FIG. 10 ). Therefore, it was demonstrated that the stem-loop structure containing two loop structures (16th to 43rd positions from the 5' end of SEQ ID NO: 4) is an important region (motif) for binding to viral proteins.

Similarly, three types of mutant aptamers (SEQ ID NO: 26 to 28) were prepared for P30-10-h1-3 (SEQ ID NO: 6) as shown in FIG. 11, and their binding inhibitory activity against H1N1pdm09 viral protein was evaluated. For P30-10-h1-3_compl1 (SEQ ID NO: 26), the 31st to 35th bases from the 5' end of P30-10-h1-3 (SEQ ID NO: 6) were mutated. Specifically, the 31st base (U) was mutated to A, the 32nd base (A) was mutated to G, the 33rd base (U) was mutated to A, the 34th base (C) was mutated to U, and the 35th base (U) was mutated to A. For P30-10-h1-3_compl2 (SEQ ID NO: 27), the 15th to 21st bases from the 5' end of P30-10-h1-3 (SEQ ID NO: 6) were mutated. Specifically, the 15th base (C) was mutated to A, the 16th base (A) to C, the 17th base (G) to U, the 18th base (A) to U, the 19th base (A) to C, the 20th base (G) to U, and the 21st base (U) to G. P30-10-h1-3_del1 (SEQ ID NO: 28) was prepared by deleting 24 bases from the 39th to 62nd positions from the 5' end of P30-10-h1-3 (SEQ ID NO: 6).
As a result, the binding inhibitory activity of P30-10-h1-3_compl2 and P30-10-h1-3_del1 was reduced ( FIG. 12 ). Therefore, it was suggested that these mutated regions are important for binding to viral proteins. In other words, the base sequence shown at 12 to 24 positions from the 5' end of SEQ ID NO:6 and the base sequence shown at 39 to 62 positions from the 5' end of SEQ ID NO:6 are predicted to be core sequences (motifs) that may be necessary for binding to viral proteins, or to contain a core sequence in part.

Claims (14)

A base sequence represented by any one of SEQ ID NOs: 4 to 11, or a base sequence in which 1 to 5 bases have been deleted, substituted or added , and the deletion, substitution or addition has been introduced within a range in which the structure of a portion of the secondary structure that has binding ability to influenza A/H1N1pdm09 virus is maintained, or the deletion, substitution or addition has been introduced at a location other than the structure of the portion.
A nucleic acid aptamer having binding ability to influenza virus A/H1N1pdm09, which comprises a nucleic acid comprising: A nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus, comprising a nucleic acid containing a base sequence shown in any one of SEQ ID NOs: 4 to 11, or a base sequence in which 1 to 5 bases have been deleted, substituted or added, and in a secondary structure formed by the nucleic acid , the secondary structure contains a base sequence that has been shortened so as to retain the structure of a portion that has binding ability to influenza A/H1N1pdm09 virus. The nucleic acid aptamer according to claim 1 or 2, wherein the nucleic acid is RNA. The nucleic acid aptamer according to any one of claims 1 to 3, wherein at least one ribose site of the nucleotide constituting the aptamer is chemically modified. The nucleic acid aptamer according to claim 4, wherein the chemical modification is modification of the 2' position of the ribose moiety with a fluoro group (2'-F) or a methoxy group (2'-OMe), or substitution with hydrogen (2'-deoxy). The nucleic acid aptamer according to any one of claims 1 to 5, wherein the 5' end and/or the 3' end are modified. A single-stranded DNA, double-stranded DNA, or RNA that contains the same base sequence as the nucleic acid aptamer described in claim 1 or 2 or a base sequence complementary to said base sequence, and that can be converted into the nucleic acid aptamer described in claim 1 or 2. A detection agent for influenza A/H1N1pdm09 virus, comprising the nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient. A diagnostic agent for influenza virus type A/H1N1pdm09, comprising the nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient. A method for detecting influenza virus type A/H1N1pdm09, comprising a step of applying a nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient to a test sample. A testing method for identifying or denying at least one of influenza virus subtypes, strains, and clades, comprising a step of applying a nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient to a test sample. A nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus,
The aptamer is
A motif consisting of the 16th to 43rd bases from the 5' end of SEQ ID NO: 4, which forms at least one loop structure;
a stem structure formed by bases added to each of the 5' end and the 3' end of the base sequence constituting the motif . The nucleic acid aptamer according to claim 12, which has an identity of 90 % or more with the base sequence shown in the 1st to 58th bases from the 5' end of SEQ ID NO:4. A nucleic acid aptamer having binding ability to influenza A/H1N1pdm09 virus,
The aptamer is
A first motif consisting of a base sequence represented by the 12th to 24th bases from the 5' end of SEQ ID NO:6 and a second motif consisting of a base sequence represented by the 39th to 62nd bases from the 5' end of SEQ ID NO: 6 ,
a loop structure formed by a base sequence including the second motif, and a stem structure formed by bases added to each of the 5'-end and 3'-end of the base sequence constituting the loop structure,
A nucleic acid aptamer having 90% or more identity to the base sequence represented by the 1st to 77th bases from the 5' end of SEQ ID NO:6.