JP2022074022A - Nucleic acid aptamer for influenza virus and detection of influenza virus - Google Patents

Abstract

To provide a nucleic acid aptamer of A/H1N1pdm09 influenza virus.SOLUTION: The present invention discloses a nucleic acid aptamer having binding affinity to A/H1N1pdm09 influenza virus, comprising a nucleic acid including a specific base sequence, or the base sequence in which one or several bases are deleted, substituted, or added in the specific base sequence.SELECTED DRAWING: Figure 4

Description

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

There are four types of influenza virus, type A, type B, type C and type D, and it is type A and type B that cause an annual epidemic in humans as seasonal influenza. Influenza A virus is classified into 144 subtypes by the combination of hemagglutinin (16 types of H1 to H16) and neuraminidase (9 types of N1 to N9), which are proteins protruding from the virus surface, and is classified into H1N1 and H2N2. , H3N2, etc.

In recent years, the influenza viruses A / H1N1pdm09 (hereinafter, also referred to as A / H1N1pdm09) and H3N2 (hereinafter, also referred to as H3N2), which were prevalent worldwide in 2009, are the causative viruses of seasonal influenza that infect humans and are prevalent. Also called H3N2). Although these influenza viruses do not show a high case fatality rate, A / H1N1pdm09 is more prominent in children and young people than H3N2, and H3N2 is relatively elderly and post-influenza. There are reports of different clinical courses for each subtype, such as the conspicuous cases of bacterial pneumonia in the case of influenza (Non-Patent Document 1). In addition, since a plurality of A subtypes may be prevalent at the same time, some patients are infected with influenza virus twice in one season (Non-Patent Document 2).
Distinguishing A subtype is important not only from the viewpoint of treatment observation but also from the viewpoint of prevention of recurrent infection and coinfection.

So far, antibodies and aptamers for discriminating subtypes have been developed, and subtype discrimination test kits using these have also been developed (Patent Document 1, Non-Patent Document 3). However, it is not always possible to discriminate epidemic strains because the epidemic subtypes differ depending on the year and region, and HA and NA, which are targets for subtype discrimination, are easily mutated because they are present on the surface of the virus.

Japanese Unexamined Patent Publication No. 2012-100636

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

Among the existing aptamers, a decrease in the binding force to the influenza virus A subtype that is prevalent is observed, and it is difficult to distinguish the subtype using these aptamers.
In order to create a diagnostic kit for discriminating influenza virus A subtype, it is necessary to continue to respond to the rapid mutation of this virus, and the development of an aptamer that responds to the current epidemic strain has been desired.

So far, a plurality of aptamers for discriminating influenza A subtype have been developed, but no aptamer that binds to the current A / H1N1pdm09 epidemic strain has been obtained.

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

The present disclosure comprises, in one embodiment, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 4 to 11 or a base sequence in which one or several bases are deleted, substituted or added. , A / H1N1pdm09 relating to a nucleic acid aptamer capable of binding to influenza virus.
The present disclosure comprises, in one embodiment, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 4 to 11 or a base sequence in which one or several bases are deleted, substituted or added. Bind ability to A / H1N1pdm09 type influenza virus consisting of nucleic acid containing a base sequence shortened so as to retain the structure of the distal region portion having binding ability to A / H1N1pdm09 type influenza virus in the secondary structure. With respect to nucleic acid aptamers having.

The present disclosure is, in one embodiment, a nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
The aptamer relates to a nucleic acid aptamer having a motif consisting of the base sequence represented by the 16th to 43rd positions from the 5'end of SEQ ID NO: 4.
The present disclosure is, in one embodiment, a nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
The aptamer consists of the first motif consisting of the 12th to 24th base sequences from the 5'end of SEQ ID NO: 6 and the 39th to 62nd base sequences from the 5'end of SEQ ID NO: 6. It relates to a nucleic acid aptamer having a second motif.

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

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

FIG. 1 is a graph showing the relationship between the number of cycles of selection and amplification of a target RNA aptamer and the amount of RNA that binds to a virus. FIG. 2 is a secondary structure (prediction) of an RNA aptamer. FIG. 3 is a secondary structure (prediction) of an RNA aptamer. FIG. 4 is a graph showing the amount of binding of the RNA aptamers of Experimental Examples 1 to 10 and Comparative Examples 1 and 2 to A / H1N1pdm09 strain Ark19907 (H1N1pdm09) and H3N2 strain Ark181002 (H3N2). FIG. 5 is a base sequence of the nucleic acid used in the examples. FIG. 6 is a base sequence of the nucleic acid used in the examples. FIG. 7 shows a mutant portion of a mutant aptamer in the secondary structure (prediction) of an RNA aptamer (P30-10-h1-1). FIG. 8 is a graph showing the amount of binding of RNA aptamer (P30-10-h1-1) and its mutant aptamer to Ark19907 (H1N1pdm09), which is an A / H1N1pdm09 strain. FIG. 9 shows a mutant portion of a mutant aptamer in the secondary structure (prediction) of an RNA aptamer (P30-10-h1-1). FIG. 10 is a graph showing the amount of binding of RNA aptamer (P30-10-h1-1) and its mutant aptamer to Ark19907 (H1N1pdm09), which is an A / H1N1pdm09 strain. FIG. 11 shows a mutant portion of a mutant aptamer in the secondary structure (prediction) of an RNA aptamer (P30-10-h1-3). FIG. 12 is a graph showing the amount of binding of RNA aptamer (P30-10-h1-3) and its mutant aptamer to Ark19907 (H1N1pdm09), which is an A / H1N1pdm09 strain.

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

[Nucleic acid aptamer]
Nucleic acid aptamers are generally nucleic acid ligands artificially created to specifically bind to viruses, proteins, peptides, saccharides, metal ions, small molecules and the like.
The nucleic acid aptamers of the present disclosure have the ability to bind to A / H1N1pdm09 influenza virus.
In the present disclosure, nucleic acids, in one or more embodiments, include DNA, RNA, and analogs thereof.
The DNA and RNA may, in one or more embodiments, contain 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 can be included in the nucleic acid aptamer of the present disclosure as a nucleic acid having a base sequence replaced so that thymine and uracil are converted, for example.

The nucleic acid aptamers of the present disclosure have, in one or more embodiments, the base sequence represented by any of SEQ ID NOs: 4 to 11, or one or several bases have been deleted, substituted or added in the base sequence. It consists of nucleic acid containing a base sequence.
The length of the nucleic acid aptamer of the present disclosure is preferably 100 bases or less in one or more embodiments, for example, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less or 40. It is below the base. 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 aptamers of the present disclosure is 20 to 80 bases or 30 to 90 bases in one or more embodiments.
The base sequences of SEQ ID NOs: 4 to 11 are obtained by a method called SELEX method, which selects a nucleic acid aptamer that strongly binds to a specific target substance. In the SELEX method, a nucleic acid aptamer that strongly binds to a target substance can be obtained by preparing a nucleic acid library having a random sequence, selecting nucleic acids bound to the target substance, and repeating the cycle of PCR amplification a plurality of times. Currently, various improved SELEX methods have been reported.
The nucleotide sequences of SEQ ID NOs: 4 to 11 are selected by a total of 10 cycles by the SELEX method, and the obtained RNA pool that binds to the specific virus is cloned. Specifically, a DNA library having 30 bases in the center of the sequence as a random region is synthesized, PCR-amplified, and then transcribed to create an RNA pool, and the RNA pool is bound to the target influenza virus. RNA was sorted and amplified. By repeating this cycle 10 times, aptamers having the base sequences shown in SEQ ID NOs: 4 to 11 having high specificity and binding (affinity) to A / H1N1pdm09 influenza virus were obtained.

[Shortened aptamer]
In general, secondary structure models of nucleic acid aptamers can be predicted. The secondary structure of the nucleic acid aptamer is a stem-loop structure consisting of a tip loop structure and a stem structure. It is considered that the site that actually binds to the target substance is mainly the stem region containing the tip loop (hereinafter, also referred to as “tip region portion”).
A secondary structure model of the nucleic acid aptamers of the present disclosure composed of RNA of the nucleotide sequences of SEQ ID NOs: 4 to 11 is shown in FIGS. 2 and 3 in one or more embodiments.
The nucleic acid aptamers of the present disclosure may include, in one or more embodiments, 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 distal region portion of the secondary structure is not destroyed.
The present disclosure comprises, in one embodiment, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 4 to 11 or a base sequence in which one or several bases are deleted, substituted or added. It has the ability to bind to the A / H1N1pdm09 type influenza virus, which consists of a nucleic acid containing a base sequence shortened so as to retain the structure of the portion having the ability to bind to the A / H1N1pdm09 type influenza virus in the secondary structure. Regarding nucleic acid aptamers.
In the present disclosure, the moiety capable of binding to A / H1N1pdm09 influenza virus is, in one or more embodiments, the distal 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 having the binding ability to A / H1N1pdm09 type influenza virus includes any of the following base sequences in one or more embodiments.
The base sequence shown at the 15th to 44th positions from the 5'end of SEQ ID NO: 4 or 7;
The base sequence shown at the 11th to 48th positions from the 5'end of SEQ ID NO: 4 or 7; the base sequence shown at the 11th to 25th positions from the 5'end of SEQ ID NO: 6;
The base sequences shown at the 6th to 28th and 38th to 72nd positions from the 5'end of SEQ ID NO: 6;
The base sequence shown at the 1st to 28th and 38th to 77th positions from the 5'end of SEQ ID NO: 6.
In the nucleic acid aptamer of the present disclosure, examples of the base sequence constituting the structure of the distal region having the ability to bind to A / H1N1pdm09 influenza virus include any of the following base sequences in one or more embodiments.
The base sequence shown at the 25th to 40th positions from the 5'end of SEQ ID NO: 4 or 7;
The base sequence shown at the 16th to 43rd positions from the 5'end of SEQ ID NO: 4 or 7;
The base sequence shown at the 15th to 44th positions from the 5'end of SEQ ID NO: 4 or 7;
The base sequence shown at the 11th to 48th positions from the 5'end of SEQ ID NO: 4 or 7;
The base sequence shown at the 11th to 25th positions from the 5'end of SEQ ID NO: 6.

The "deletion, substitution or addition" of one or several bases in the nucleic acid aptamers of the present disclosure is, in one or more embodiments, the structure of a portion of the secondary structure capable of binding to A / H1N1pdm09 influenza virus. It is introduced within the range that holds the virus, or it is introduced in a place other than the structure of the same part.
In the present disclosure, the number is 2, 3, 4, or 5 in one or more embodiments.

Nucleic acid aptamers comprising deletions or substitutions of bases in the base sequences of SEQ ID NOs: 4 to 11 are 10% or more, 20% or more, in one or more embodiments, as compared to nucleic acid aptamers having the corresponding base sequences. It may have 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 substantially the same or the same level of the above-mentioned binding ability. Nucleic acid aptamers comprising deletions or substitutions of bases in the base sequences of SEQ ID NOs: 4 to 11 have a higher level of binding ability than nucleic acid aptamers having the corresponding base sequences in one or more embodiments. It may also have, for example, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more higher levels of the above-mentioned binding ability.

The binding ability of the nucleic acid aptamers of the present disclosure can be evaluated in one or more embodiments by the amount of A / H1N1pdm09 binding to influenza virus relative to the amount of binding to H3N2 (Ark181002) influenza virus. The amount of A / H1N1pdm09 bound to influenza virus relative to the amount of bound H3N2 (Ark181002) influenza virus in the nucleic acid aptamers of the present disclosure is 3 times or more, 5 times or more, 10 times or more, 20 in one or more embodiments. It is fold 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 nucleic acid aptamer bound to influenza virus can be measured by the RT-qPCR method. Specifically, it can be measured by the method described in Examples.

[Other aspects of shortened aptamer 1]
In the nucleic acid aptamer formed by the base sequence represented by SEQ ID NO: 4, the present inventor presents that the base sequence represented by the 16th to 43rd positions from the 5'end of SEQ ID NO: 4 has the binding property to A / H1N1pdm09 influenza virus. Found to be involved in. Therefore, the nucleic acid aptamer of the present disclosure is, as another embodiment, a nucleic acid aptamer having a binding ability to A / H1N1pdm09 type influenza virus, and is a base represented by the 16th to 43rd positions from the 5'end of SEQ ID NO: 4. It has a motif consisting of an array.

The motif of this embodiment may have one or more loop structures in one or more embodiments without particular limitation, preferably two loop structures and one stem located between the two loop structures. It may have a structure. The nucleic acid aptamer of this embodiment has, in one or more embodiments, a structure formed by two loop structures and one stem structure located between the two loop structures.

In the present disclosure, the "loop structure" refers to a single-stranded loop-like (cyclic) structure that does not form a base pair in a single-stranded nucleic acid. In one or more embodiments, the loop structure can also be said to be a loop-like structure that is located between the double strands forming the stem structure and does not form a base pair. The number of bases forming the loop structure is 5 or more and 50 or less (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) in one or more embodiments. 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 "stem structure" refers to a chain-like structure formed by forming a base pair of one or more complementary bases of a single-stranded nucleic acid. In one or more embodiments, the stem structure may be a chain structure formed by forming a base pair of a part or consecutive bases of a constituent base completely or partially with each other. good. The number of bases forming the stem structure is 2 or more and 20 or less (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, in one or more embodiments). 15, 16, 17, 18, 19 or 20).
The stem structure of the nucleic acid aptamer of this embodiment comprises, in one or more embodiments, two consecutive GC base pairs.

In one or more embodiments, the nucleic acid aptamer of this embodiment may have two or more bases 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 embodiment comprises the motif forming at least one loop structure and the bases added to the 5'end and 3'end of the base sequence constituting the motif, respectively. Including the stem structure formed by. The number of bases forming the loop structure is 5 or more and 20 or less (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) in one or more embodiments. , 19 or 20).
One or more embodiments of the nucleic acid aptamer of the embodiment without particular limitation include nucleic acids containing any of the following base sequences.
The base sequence shown at the 15th to 44th positions from the 5'end of SEQ ID NO: 4;
The base sequence shown at the 14th to 45th positions from the 5'end of SEQ ID NO: 4;
The base sequence shown at the 13th to 46th positions from the 5'end of SEQ ID NO: 4;
The base sequence shown at the 12th to 47th positions from the 5'end of SEQ ID NO: 4;
Nucleotide sequence represented by the 11th to 48th positions from the 5'end of SEQ ID NO: 4 One or more embodiments of the nucleic acid aptamer of the embodiment are 16th to 43rd from the 5'end of SEQ ID NO: 4. Examples thereof include nucleic acids having a motif consisting of the indicated base sequence and containing a base sequence having 85% or more identity with any of the above five sequences. The aptamer retains the base sequence of the above motif in one or more embodiments.

In one or more embodiments, the nucleic acid aptamer of this embodiment has the above-mentioned motif and has 85% or more identity with the base sequence represented by the 1st to 58th positions from the 5'end of SEQ ID NO: 4. .. The nucleic acid aptamer of this embodiment, in one or more embodiments without particular limitation, has one or more loop structures and one or more stem structures, preferably two loop structures and two loop structures formed by the motif. It may have one stem structure located between the loop structures and a stem structure located at the 5'and 3'ends of the motif.

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

[Other aspects of shortened aptamer 2]
In the nucleic acid aptamer formed by the base sequence represented by SEQ ID NO: 6, the present inventor has the base sequence represented by the 12th to 24th from the 5'end of SEQ ID NO: 6 and the 39th from the 5'end of SEQ ID NO: 6. It was found that the nucleotide sequence shown in the 62nd to 62nd is involved in the binding to the A / H1N1pdm09 type influenza virus. Therefore, the nucleic acid aptamer of the present disclosure is, as another embodiment, a nucleic acid aptamer having a binding ability to A / H1N1pdm09 type influenza virus, and is a base represented by the 12th to 24th positions from the 5'end of SEQ ID NO: 6. It has a first motif consisting of a sequence and a second motif consisting of the base sequences represented by the 39th to 62nd positions from the 5'end of SEQ ID NO: 6.

The first motif has a stem-loop structure in one or more embodiments without particular limitation. The base forming the loop structure is 5 or more and 10 or less (for example, 5, 6, 7, 8, 9 or 10) in one or more embodiments.

The nucleic acid aptamer of this embodiment may have, in one or more embodiments, a loop structure formed by a base sequence containing the second motif. The nucleic acid aptamer of this embodiment, in one or more embodiments, has 2 or more and 20 or less (eg, 2, 3, 4, 5, etc.) at each of the 5'end and 3'end of the base sequence forming the loop structure. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) bases may be added. In one or more embodiments, the added base may partially or wholly form a base pair to form a stem structure.

In one or more embodiments, the nucleic acid aptamer of this embodiment has a stem-loop structure formed by the first motif, a loop structure formed by a base sequence containing the second motif, and the loop structure. The stem-loop structure formed by the first motif contains the second motif, and includes the stem structure formed by the bases added to each of the 5'end and the 3'end of the base sequence constituting the above. It is linked via a stem structure and a loop structure formed by a base sequence.
One or more embodiments of the nucleic acid aptamer of the embodiment without particular limitation include nucleic acids containing any of the following base sequences.
The base sequences shown at the 6th to 28th and 38th to 72nd positions from the 5'end of SEQ ID NO: 6;
The base sequences shown at the 5th to 28th and 38th to 73rd positions from the 5'end of SEQ ID NO: 6;
The base sequences shown at the 4th to 28th and 38th to 74th positions from the 5'end of SEQ ID NO: 6;
The base sequence shown at the 3rd to 28th and 38th to 75th positions from the 5'end of SEQ ID NO: 6;
The base sequences shown at the 2nd to 28th and 38th to 76th positions from the 5'end of SEQ ID NO: 6;
The base sequence shown at the 1st to 28th and 38th to 77th positions from the 5'end of SEQ ID NO: 6.
One or more embodiments of the nucleic acid aptamer of the embodiment are not particularly limited, having the first motif and the second motif described above, and having 85% or more identity with any of the six sequences described above. Nucleic acid containing a base sequence can be mentioned. The aptamer retains the base sequences of the first and second motifs described above in one or more embodiments.

In one or more embodiments, the nucleic acid aptamer of this embodiment has the above-mentioned first motif and second motif, and has the base sequence represented by the 1st to 77th positions from the 5'end of SEQ ID NO: 6. Has an identity of 85% or more with respect to. The nucleic acid aptamer of the embodiment has one or more loop structures and one or more stem structures, preferably a loop structure containing the first motif, and the loop structure in one or more embodiments without particular limitation. It may have a stem-loop structure formed by a second motif connected to the first motif, and a stem structure located at the 5'end and 3'end of the loop structure containing the first motif.

[Chemical modification]
The nucleic acid aptamers of the present disclosure may include chemically modified embodiments as described above.
When the nucleic acid is RNA, it may have a chemically modified ribonucleotide in the base sequence of the nucleic acid aptamer in order to add ribonuclease resistance. Such a nucleic acid aptamer may, for example, change the 2'-OH group of the ribose moiety of the ribonucleotide in the nucleic acid aptamer to a fluoro group (2'-F) or a methoxy group (2'-OMe) by a conventional method, or It is obtained by substituting the 2'position of the ribose moiety with hydrogen (2'-deoxy).
These chemical modifications are more effective against pyrimidine nucleotides because the pyrimidine nucleotide sites are more susceptible to degradation by ribonucleases.
These chemical modifications are introduced in one or more embodiments in a secondary structure other than the structure of the moiety capable of binding to A / H1N1pdm09 influenza virus. These chemical modifications are made to the ribose group of the pyrimidine nucleotide in the loop region in one or more embodiments.

The chemical modification may be, in one or more other embodiments, modification with 5'end and / or 3'end of the nucleic acid aptamer with inverted deoxythymidine (idT) or polyethylene glycol (PEG). These improve the ribonuclease resistance of aptamer RNA.
Since the decrease in activity due to decomposition in a living body can be suppressed by chemical modification, the nucleic acid aptamer of the present disclosure can be used as a pharmaceutical composition exhibiting an antiviral action in the living body.
The present disclosure relates to, in other embodiments, a pharmaceutical composition comprising the nucleic acid aptamer of the present disclosure as an active ingredient, which exerts an antiviral effect on A / H1N1pdm09 influenza virus.
In the present disclosure, "using the nucleic acid aptamer of the present disclosure as an active ingredient" may include, in one or more embodiments, a form containing an intermediate described later.

[Marking body]
The nucleic acid aptamers of the present disclosure can be used in one or more embodiments for the detection of A / H1N1pdm09 influenza virus, as described below.
The nucleic acid aptamers of the present disclosure include labeled embodiments for detection.
The label can be appropriately selected depending on the detection method, and in one or more embodiments, fluorescent dyes, digoxigenin, digoxin, biotin, radioactive substances and the like can be mentioned.

[Intermediate]
The present disclosure relates, in one aspect, to intermediates that can be converted in vivo or in vitro to the nucleic acid aptamers of the present disclosure.
The intermediate contains the same base sequence of the base sequence of the nucleic acid aptamer of the present disclosure or a base sequence complementary to the base sequence, and is disclosed by an in vitro gene manipulation means or an in vivo or intracellular reaction. Examples include single-stranded DNA, double-stranded DNA, and RNA that can be converted to nucleic acid aptamers of. These DNAs and RNAs may include these chemically modified forms, and combinations thereof.
The present disclosure, in one embodiment, is a single-stranded DNA, double-stranded DNA, which comprises 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. Regarding DNA or RNA.

[Production method]
The aptamers of the present disclosure can be produced by chemical synthesis based on a base sequence in one or more embodiments. The DNA aptamer can be chemically synthesized from a terminal base using dNTP as a material by using a DNA synthesizer. Protection of the 2'hydroxyl group at the ribose site is required for RNA synthesis, and various amidites have been developed as protecting groups, and 2-cyanoethoxymethyl (CEM) groups can be used.
In one or more embodiments, the aptamer of the present disclosure chemically synthesizes the above DNA corresponding to the RNA aptamer for synthesis, PCR-amplifies the DNA, and synthesizes the RNA aptamer from the amplified DNA by a transcription reaction with an RNA polymerase. It can be produced by the method of (in vitro transcription method).
The aptamers of the present disclosure can also be obtained from complementary RNA in one or more embodiments by using an RNA-dependent RNA polymerase.

[Detection method]
The nucleic acid aptamers of the present disclosure can be used in one or more embodiments for the detection of A / H1N1pdm09 influenza virus, as described below.
The present disclosure relates to a method for detecting A / H1N1pdm09 influenza virus (hereinafter, also referred to as "the detection method of the present disclosure"), which comprises a step of allowing the nucleic acid aptamer of the present disclosure to act as an active ingredient on a sample to be inspected. .. The detection method of the present disclosure is, in one or more embodiments, contacting the nucleic acid aptamer of the present disclosure with a sample to be inspected, and binding of the nucleic acid aptamer of the present disclosure to the A / H1N1pdm09 influenza virus in the sample. Including measuring. Alternatively, the detection method of the present disclosure is to bring the nucleic acid aptamer of the present disclosure into contact with a sample to be inspected, and to measure whether or not the nucleic acid aptamer of the present disclosure binds to the A / H1N1pdm09 influenza virus in the sample. including.
Acting as an active ingredient includes, in one or more embodiments, use as an indicator of detection, binding to A / H1N1pdm09 influenza virus, or as a major factor in the detection mechanism.
In the present disclosure, detection of a virus includes detecting the virus in a sample, and confirming that the virus is present or absent in a sample suspected of containing the virus. include.
If the presence or absence of the virus can be confirmed in the sample, at least one of the influenza virus subtypes, strains, and clades (strains) contained in the sample can be identified or denied.
The present disclosure relates to a test method for identifying or denying at least one of influenza virus subtypes, strains, and clades, comprising the step of allowing the nucleic acid aptamer of the present disclosure to act as an active ingredient on a sample to be tested. The testing methods of the present disclosure, in one or more embodiments, bring the nucleic acid aptamer of the present disclosure into contact with a sample to be tested and the binding of the nucleic acid aptamer of the present disclosure to the A / H1N1pdm09 influenza virus in the sample. Including measuring. Alternatively, the test method of the present disclosure is to bring the nucleic acid aptamer of the present disclosure into contact with a sample to be inspected, and to measure whether or not the nucleic acid aptamer of the present disclosure binds to the A / H1N1pdm09 influenza virus in the sample. including.

As the sample to be inspected in the present disclosure, in one or a plurality of embodiments, a sample collected from a subject's nasal cavity, pharyngeal swab, nasal discharge, etc., or the sample is given to fertilized chicken eggs, cultured cells, etc. to infect and proliferate. Examples thereof include the obtained solution. Examples of the sample to be inspected include, in one or more embodiments, a sample containing 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 fluorosane, rhodamine, Texas red, etc. is brought into contact with a test sample to carry out a binding reaction, and after removing the unbound nucleic acid aptamer, fluorescence or its intensity is detected. / Or by measuring, the detection of A / H1N1pdm09 type influenza virus or the amount thereof can be measured. For example, it can be carried out using a substrate on which either a labeled aptamer RNA or a test sample is immobilized.
Further, as a method of not immobilizing either the labeled aptamer or the test substance, a substance that fluoresces at the time of binding is used to label either the aptamer or the test sample of the present disclosure, and the fluorescence at the time of binding or its intensity is determined. It can also be performed by detection and measurement. Examples of such fluorescent dyes include fluoroscein, rhodamine, Texas red and the like.

Detection Method Using Surface Plasmon Resonance Method (SPR) As one or more embodiments of the detection method of the present disclosure, a method of using the nucleic acid aptamer of the present disclosure without a fluorescent label will be described.
In the SPR method, a minute mass change caused by the binding of a molecule immobilized on the sensor chip and a molecule passing on the sensor chip can be detected as a change in the refractive index of the reflected light. By immobilizing either the aptamer RNA or the target substance of the present disclosure on the surface of a gold thin film on a sensor chip by a well-known method and supplying the test substance or the aptamer RNA to the surface, the binding reaction of these molecules can be measured in real time. do.
As an apparatus using the SPR method, for example, there is Biacore T100 manufactured by GE Healthcare Bioscience.

Detection method using an immunochromatographic method As one or more embodiments of the detection method of the present disclosure, a method using an immunochromatographic method will be described.
As one form, there is an immunochromatographic method using the nucleic acid aptamer of the present disclosure as a capture substance for fixing the virus at the detection site of the test piece (support) on which the sample is developed.
One form includes an immunochromatography method using the nucleic acid aptamer of the present disclosure as a capture substance for binding to and labeling a virus immobilized on a detection site of a test piece (support).
As one form, one or two kinds of nucleic acid aptamers of the present disclosure are used as a capture substance for fixing a virus at the detection site of a test piece (support) on which a sample is developed, and a test piece (support). Examples thereof include an immunochromatography method used as a capture substance for binding to and labeling a virus immobilized on a detection site.
The nucleic acid aptamers of the present disclosure used as a capture substance for labeling can use the nucleic acid aptamers of the present disclosure in labeled embodiments in one or more embodiments.

[Diagnosis method]
The detection method and the test method of the present disclosure can be used for a method for diagnosing influenza.
The present disclosure, in other embodiments, confirms whether or not the A / H1N1pdm09 influenza virus is present in the sample to be inspected by the detection method or the inspection method of the present disclosure, and provides the sample from the results. The present invention relates to a diagnostic method including determining whether or not the subject is infected with the virus.

[Detective, diagnostic agent, kit]
The present disclosure relates, in one aspect, to a detection agent, a diagnostic agent, or a kit containing the nucleic acid aptamer of the present disclosure as an active ingredient for use in the detection method, the inspection method, or the diagnostic method of the present disclosure.
The detection agent can be used in the detection method and the inspection method 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 companion diagnostic agents for therapeutic agents against the A / H1N1pdm09 influenza virus.
The kit can be used for the detection method, the inspection method, and the diagnostic method of the present disclosure.
In the production of detection agents, diagnostic agents, and kits, they are appropriately used in combination with well-known pharmaceutically acceptable diluents, stabilizers, other carriers, and the like. In addition to the nucleic acid aptamers disclosed in the present disclosure, these may contain reagents and test pieces used for detection.

The present disclosure may relate to one, but not limited to, one or more embodiments:
[1] A / H1N1pdm09 type consisting of the base sequence represented by any of SEQ ID NOs: 4 to 11 or a nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the base sequence. Nucleic acid aptamer capable of binding to influenza virus.
[2] In the base sequence represented by any of SEQ ID NOs: 4 to 11, or in a secondary structure composed of a nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the base sequence. A nucleic acid aptamer having a binding ability to A / H1N1pdm09 type influenza virus, which comprises a nucleic acid containing a base sequence shortened so as to retain the structure of a portion having a binding ability to A / H1N1pdm09 type influenza virus.
[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 ribose site of the nucleotide constituting the aptamer is chemically modified.
[5] The chemical modification is a modification with a fluoro group (2'-F) or a methoxy group (2'-OMe) for the 2'position of the ribose moiety, or a substitution with hydrogen (2'-deoxy) [4]. ] The nucleic acid aptamer according to.
[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] Contains the same base sequence as the nucleic acid aptamer according to any one of [1], [2] and [12] to [16], or a base sequence complementary to the base sequence, and [1], [ 2] and a single-stranded DNA, double-stranded DNA or RNA that can be converted into the nucleic acid aptamer according to any one of [12] to [16].
[8] A detection agent for A / H1N1 pdm09 influenza virus containing the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] as an active ingredient.
[9] A diagnostic agent for A / H1N1 pdm09 influenza virus containing the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] as an active ingredient.
[10] A method for detecting influenza A / H1N1pdm09, which comprises a step of allowing the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] to act as an active ingredient on a sample to be inspected.
[11] Influenza virus subtypes, strains, and clades comprising the step of allowing the nucleic acid aptamer according to any one of [1] to [6] and [12] to [16] to act as an active ingredient on the sample to be inspected. An inspection method that identifies or denies at least one of.
[12] A nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
A nucleic acid aptamer in which the aptamer has a motif consisting of the base sequences represented by the 16th to 43rd positions from the 5'end of SEQ ID NO: 4.
[13] The motif forms at least one loop structure and forms.
[12] The nucleic acid aptamer according to [12], wherein the nucleic acid aptamer further has a stem structure formed by a base added to each of the 5'end and the 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 shown at positions 1 to 58 from the 5'end of SEQ ID NO: 4.
[15] A nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
The aptamer consists of the first motif consisting of the 12th to 24th base sequences from the 5'end of SEQ ID NO: 6 and the 39th to 62nd base sequences from the 5'end of SEQ ID NO: 6. Nucleic acid aptamer with a second motif.
[16] The aptamer has a loop structure formed by a base sequence containing the second motif, and is formed by bases added to the 5'end and 3'end of the base sequence constituting the loop structure, respectively. [15] The nucleic acid aptamer according to [15], which further has a stem structure.
[17] The nucleic acid aptamer according to [15] or [16], which has 85% or more identity with respect to the base sequence represented by the 1st to 77th positions from the 5'end of SEQ ID NO: 6.

Hereinafter, the present disclosure will be described in more detail by way of examples, but these are exemplary and the present disclosure is not limited to these examples.

1. 1. In vitro selection of influenza virus A (H1N1) pdm09-specific aptamer (1-1) Acquisition and establishment of clinical isolates (1-1-1) Collection of samples from influenza-infected persons 2018/2019 season and 2019 In the 2020 season, the nasal cavity of a patient suspected of having influenza infection was wiped with a special swab, or nasal juice was collected, and the presence or absence of infection was determined using the influenza antigen detection kit SPOTCHEM FLORA FluAB manufactured by Arcley. If influenza A is positive, the patient's nasal juice is collected with the swab included in the kit, and 500 to 1000 μl of VTM (Copan) or antibiotic-antifungal mixture (Nakalitesk, hereinafter antibiotic) is added. It was suspended in Dulvecco's Modified Eagle Medium (manufactured by Sigma-Aldrich).
(1-1-2) Isolation and culture of influenza virus Madin-Darby Canine Kidney (MDCK) cells were seeded on a 12-well plate so as to be 100% confluent the next day, and cultured overnight at 37 ° C. in a 5% CO ₂ environment. bottom. The next day, the supernatant of 100% confluent cells was removed and washed twice with 500 μl PBS (−). 200 μl of a diluted sample collected from an influenza-infected person was added to MDCK cells and incubated for 30 minutes in a 5% CO ₂ environment at 34 ° C. to adhere the virus to the cell surface. 800 μl of DMEM medium supplemented with an antibiotic and 2.5 μg / ml of acetyltrypsin (manufactured by Sigma-Aldrich) was added, and the cells were cultured in a 34 ° C. 5% CO ₂ environment for 5 to 7 days.
10 to 50 μl of the culture supernatant was collected for each culture day, measured with an influenza antigen detection kit (manufactured by ARKRAY, Inc.), the amount of virus in the culture supernatant was monitored, and the type of influenza was discriminated.
(1-1-3) Subtype discrimination of clinical isolates, base sequence analysis of hemagglutinin gene The entire culture supernatant of cells with increased viral load was collected, centrifuged at 3000 rpm, and the supernatant was recovered (virus). solution). Of these, the RNA of influenza virus contained in 140 μl of the supernatant was extracted using a virus RNA extraction kit (manufactured by Qiagen). Subtype discrimination was performed on the extracted RNA by the real-time PCR method. For sequence analysis of the hemaglutinine gene, the viral RNA extracted by SuperScript (trade name) III One-Step RT-PCR System with Platinum Taq DNA Polymerase was reverse-transcribed, and the total length of the hemagglutinin gene was amplified by PCR. It was confirmed that a fragment of the target size was amplified by agarose gel electrophoresis, and the base sequence analysis of this fragment was performed. Real-time PCR, cloning, primers used for sequencing, and protocols were performed with reference to the 4th edition of the Influenza Diagnostic Manual.
The resulting clinical isolate was obtained from the HA gene phylogenetic tree Clade 6B. A virus strain of H1N1pdm09 influenza virus belonging to 1A (hereinafter, also referred to as [Ark19907 (H1N1pdm09)]) and Clade 3C. It was confirmed that it is a virus strain of H3N2 influenza virus belonging to 2a (hereinafter, also referred to as [Ark181002 (H3N2)]).

The selection of nucleic acid aptamers by the SELEX method shown in (1-2) to (1-4) below was carried out by modifying the conditions with reference to the 4th edition of the Influenza Diagnosis Manual.
(1-2) Creation of RNA random pool The library of single-stranded DNA (ssDNA) with the central 30 bases as the random region shown below was synthesized and used as a template (SEQ ID NO: 1), and the 5'end primer (SEQ ID NO: 1). PCR was performed using SEQ ID NO: 2) and 3'end primer (SEQ ID NO: 3).
SEQ ID NO: 1: AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG- (N) 30-CCTTTTCCTCTTCCTTCCTCTCT
SEQ ID NO: 2: AGTAATACGACTCACTATAGGGAGAATTCCGACCAGAAG
SEQ ID NO: 3: AGAAGAGGAAGGAGAGAGAGAAAGG
Transcription was then performed in vitro using a T7 Amplifier kit (manufactured by Epicenter Technologies) to convert the amplified DNA library into an RNA library.

(1-3) Sorting in vitro The RNA library (10 μg = 4 ³⁰ ≈ 1.15 × 10 ¹⁸ different RNA sequences) obtained in (1-2) above was used as a binding buffer (0.01M HEPES, 0). It was dissolved in .15 M NaCl, pH 7.4). To promote equilibrium of RNA conformation, it was treated at 95 ° C. for 2 minutes for denaturation and then cooled at room temperature for 10 minutes. In order to remove RNA that binds to the target non-specifically, tRNA (total tRNA of E. coli (manufactured by Roche)) was added to the RNA library solution as a competitor, and then the target virus solution was added. In each sorting cycle, a negative selection was first performed using Ark181002 (H3N2) as the countervirus. RNA that did not bind to this virus was recovered and then reacted with Ark19907 (H1N1pdm09) for positive selection. The molecular ratios of RNA (RNApool), viral protein (Counter / target) and tRNA (Competitor) used in each selection cycle are as shown in Table 1.

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

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

(1-4) Sorting method and amplification cycle In order to obtain an RNA aptamer having specificity and high affinity for influenza virus, the amount of RNA and viral protein was changed for each sorting cycle as shown in Table 1. In order to avoid enrichment of non-specifically bound RNA, in each of the second, fourth, sixth, eighth, ninth and tenth sorting cycles, a 96-well titer plate (manufactured by Thermo Fisher) instead of a filter. It was used. For selection on the plate, 100 μg of the above viral protein was first immobilized in each well per 1 ml of borate buffer at pH 8.0 and blocked with BSA (1% stock solution). Each well was then washed and used for sorting.
The RNA pool obtained in the previous cycle was denatured in Binding buffer at 95 ° C. for 2 minutes. Subsequently, after cooling at room temperature for 10 minutes, tRNA was added and added to the well on which the viral protein was immobilized. After incubating for 10 minutes, with 300 μl of Binding buffer (4 times in the 2nd and 3rd sorting cycles, 6 times in the 6th and 8th sorting cycles, 8 times in the 9th and 10th sorting cycles). Washed to remove unbound RNA. Then, the viral protein-binding RNA was recovered with heated Elution buffer (0.01M HEPES, 0.15M NaCl, 7M Urea, pH 7.4), precipitated and purified with ethanol, and then subjected to reverse transcription, PCR, and in vitro transcription. Replayed.

(1-5) Evaluation of RNA enrichment To evaluate the progress of enrichment of high-affinity aptamers and their specificity, RNA pool binding in the 0th, 1st, 5th, and 10th sorting cycles. The activity was analyzed by the filter binding quantification method (RT-qPCR method). RNA pools for each sorting cycle were prepared and combined with Ark19907 (H1N1pdm09) and Ark181002 (H3N2) in solution. The binding reaction was carried out by adding Escherichia coli tRNA having a molar concentration of 10 times excess as a non-specific competition inhibitor, and mixing 50 nM RNA with 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 RNA bound to the virus was trapped on the filter and washed with 2 ml of binding buffer. The viral protein-binding RNA captured on the filter was immersed in 200 μl of Elution buffer, heated to elute, and the RNA was recovered by the ethanol precipitation method. 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 (manufactured by TakaraBio), and Primescript attached buffer. 10 μl of Real-time PCR reagent PowerSYBR Green Master Mix (manufactured by Thermo Fisher) was mixed with 1 μl of 5 μM forward primer, 1 μl of 5 μM reverse primer, and 9 μl of cDNA, and qPCR was performed using the reverse transcript as a template. The binding activity was expressed by the ratio of the RNA binding amount of the RNA pool in each sorting cycle when the binding amount of the RNA pool to the virus in the 0th sorting cycle was 1. (FIG. 1). The proportion of RNA captured by the filter after the 10th sorting cycle was 7.0 times for RNA bound to Ark19907 (H1N1pdm09) and 2.0 times for RNA bound to Ark181002 (H3N2).

(1-6) Analysis of aptamers In order to obtain individual aptamers, the PCR product obtained in the 10th sorting cycle was introduced into a TA cloning vector (manufactured by Invitrogen) and transformed into Escherichia coli. Individual plasmid DNA was isolated with a plasmid purification kit (manufactured by Promega), the DNA base sequence was decoded, and the RNA base sequence corresponding to the DNA base sequence was obtained. The sequenced RNA clones were classified into a total of 10 different sequences.

P30-10-h1-1: GGGAGAAUCCGACCAGAAGUAGCUAGCCCGGGUGUGUGGUUUAUGGUCGCCCCCUUCUCUCUCUCUCUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU
P30-10-h1-2: GGGAGAAUUCGACCAGGAGGCGCGGAUGUGUGGUUGUGGUGGGUGGGGCGCGCCCUUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU
Ｐ３０－１０－ｈ１－３：ＧＧＧＡＧＡＡＵＵＣＣＧＡＣＣＡＧＡＡＧＵＧＵＣＧＡＵＧＵＧＵＡＵＣＵＵＡＵＵＵＧＵＵＵＧＵＵＵＧＵＵＵＧＵＵＵＧＵＵＵＧＵＣＣＵＵＵＣＣＵＣＵＣＵＣＣＵＵＣＣＵＣＵＵＣＵ（配列番号６）
P30-10-h1-4: GGGAGAAUUCGACCAGAAGGCUAUUGGGUGUGUGUGUGCUGUAUUGGGGUGGGUGCUCUUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCCUUGCGGUUGGUGUGUAUUGGGUGUGGGUGCCUUCUCUCUCUCUCUCUCCUCUCU
P30-10-h1-5: GGGAGAAUUCGACCAGAUCCCCUCCUCCGUAUUCGUAUGUGCGUUGCCCUUCCUCUCUCCUCUCUCUCUCUCUCUCUCUCUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU
P30-10-h1-6: GGGAGAAUUCGACCAGAAGUAAGCUAGCCCGGGUGUGUGGUUUAUGGCCGCCCCUUCCUCUCUCUCCUCUCUCUCUCU (SEQ ID NO: 9)
P30-10-h1-7: GGGAGAAUUCGACCAGAAGUAAGCUAGCCCGGGUGUGUGGUUUAUGGUCCGUCCCUUCCUCUCUCCUCUCUCUCUCU (SEQ ID NO: 10)
P30-10-h1-8: GGGAGAAUUCGACCAGAAGCGCGCGAUUGUGUGUGUGGUGGGUGGGGCGCGCCCUUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU
P30-10-h1-9: GGGAGAAUUCGACCAGAAGGAACAUUUGUGGGUGUGUGUGUGGUGGCUGUUCCUUCUUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCAUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU
Ｐ３０－１０－ｈ１－１０：ＧＧＧＡＧＡＡＵＵＣＣＧＡＣＣＡＧＡＡＧＧＧＵＣＧＧＵＧＵＡＵＡＡＵＵＧＵＡＧＵＵＵＵＧＵＵＧＵＵＧＵＵＧＵＵＧＵＵＧＵＵＧＣＣＵＵＵＣＣＵＣＵＣＵＣＣＵＵＣＣＵＣＵＵＣＵ（配列番号１３）

Aptamer P30-10-h1-1 (SEQ ID NO: 4) occupies 63.5% of the total RNA base sequence selected by targeting Ark19907 (H1N1pdm09), 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) account for 3.8% of the total, respectively, P30-10-h1-5 to P30-10-h1-10 (SEQ ID NO: 7). SEQ ID NOs: 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 by only one base from P30-10-h1-1. Secondary structure of aptamer obtained using the secondary structure analysis software "The mfold Web Server" (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form can be analyzed) I made a prediction. As for the secondary structure, a plurality of structures may be predicted for each aptamer RNA, but the structure predicted to be the most stable for each aptamer RNA is shown (FIGS. 2 and 3).

2. 2. Affinity analysis of aptamers and each viral protein by filter binding quantification method (RT-qPCR method) Above 1. The binding efficiency of the 10 aptamers selected in 1 and the influenza virus clinical isolate Ark19907 (H1N1pdm09) was determined by the filter binding quantification method (1-5 described above) (Comparative Examples 1 and 2 and Experimental Examples 1 to 10). .. The results are shown in Table 2 and FIG.
As a comparative example, D-12 (SEQ ID NO: 14) and D-26 (SEQ ID NO: 15), which are known aptamers specific for hemagglutinin of A / California / 07/2009 (H1N1) pdm09 influenza virus, were used (specially. Open 2012-100636). 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: GGAGCUCAGCCUUCACUGCCAAAGUGCCGAGGCAGUGUGUGGUGCUGUCCUCGAGUUCUAAAGUCGUUAGGAAGCGCAGCCUCAACAUGUUAACAGGCACCGUCCGUCCGUACC
Aptamer D26: GGAGCUCAGCCUUCAUGCCAAAAAAGUGCGCCAGCAAAAUGCGAGGCUGAUCCGGUGACUGGCUACAGGAGGCCUGUCCACCGUCGGAUCC (SEQ ID NO: 15)

Aptamar 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) had a larger amount of binding to Ark19907 (H1N1pdm09) than Ark181002 (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- It was shown that 10-h1-7 and P30-10-h1-8 are aptamers with stronger binding force compared to the existing aptamers D12 and D26 (2'-F). The amount of aptamer RNA binding to Ark19907 (H1N1pdm09) and Ark181002 (H3N2) was as shown in Table 2.

<Evaluation of mutant aptamer binding>
In order to identify the sequence required for binding of the aptamer to the target substance, as shown in FIG. 6, a mutant aptamer in which a partial sequence was deleted, inserted or mutated was prepared, and the binding property with Ark19907 (H1N1pdm09) was evaluated. bottom.
Specifically, biotin-modified aptamers (P30-10-h1-1 (SEQ ID NO: 4) and P30-10-h1-3 (SEQ ID NO: 6)) obtained by the SELEX method were used, and this biotin was used. The binding ability of the modified aptamer to the viral protein was evaluated by measuring the extent to which the binding of the modified aptamer to the viral protein was inhibited by the addition of the unlabeled mutant aptamer.

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

Since these refer to two loop structures, as shown in FIG. 9, six types of mutant aptamers in which mutations were added to the base of the region connecting the two loop structures were prepared (SEQ ID NOs: 20 to 25). In the prepared mutant aptamers, del1 and del2 are defective (the base in the square frame in FIG. 9 is deleted), in1 and in2 are inserted (inserted at the arrow points in FIG. 9), and mt1 and mt2 are point mutants (inserted at the arrow points in FIG. 9). It is a mutant aptamer (replacement of the base in the square frame of FIG. 9). Specifically, P30-10-h1-1_del1 (SEQ ID NO: 20) lacks the 27th base (C) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4), and the 38th base (C) is deleted. The base (G) was deleted. P30-10-h1-1_del2 (SEQ ID NO: 21) lacks the 25th and 26th bases (AG) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4), and the 38th and 39th bases. (GU) was deleted. P30-10-h1-1_in1 (SEQ ID NO: 22) is located between the 27th base (C) and the 28th base (C) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4). One base (A) was inserted, and one base (A) was inserted between the 37th base (G) and the 38th base (G). P30-10-h1-1_in2 (SEQ ID NO: 23) is located between the 27th base (C) and the 28th base (C) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4). Two bases (CC) were inserted, and two bases (GG) were inserted between the 37th base (G) and the 38th base (G). P30-10-h1-1_mt1 (SEQ ID NO: 24) mutated the 39th base (U) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4) to C. P30-10-h1-1_mt2 (SEQ ID NO: 25) mutated the 27th base (C) from the 5'end of P30-10-h1-1 (SEQ ID NO: 4) to A.
As a result of evaluating the binding inhibitory activity of these mutant aptamers on the H1N1pdm09 type viral protein, no binding inhibitory activity was shown in any of the mutant aptamers (FIG. 10). Therefore, it was shown that the stem-loop structure containing the two loop structures (16th to 43rd from the 5'end of SEQ ID NO: 4) is an important region (motif) for binding to the viral protein.

Similarly, for P30-10-h1-3 (SEQ ID NO: 6), three types of mutant aptamers (SEQ ID NOs: 26 to 28) were prepared as shown in FIG. 11 and their binding inhibitory activity against the H1N1pdm09 type viral protein was evaluated. P30-10-h1-3_compl1 (SEQ ID NO: 26) mutated the 31st to 35th bases from the 5'end of P30-10-h1-3 (SEQ ID NO: 6). Specifically, the 31st base (U) is A, the 32nd base (A) is G, the 33rd base (U) is A, and the 34th base (C) is U, 35. The second base (U) was mutated to A. P30-10-h1-3_compl2 (SEQ ID NO: 27) mutated the 15th to 21st bases from the 5'end of P30-10-h1-3 (SEQ ID NO: 6). Specifically, the 15th base (C) is A, the 16th base (A) is C, the 17th base (G) is U, and the 18th base (A) is U, 19. The second base (A) was mutated to C, the 20th base (G) to U, and the 21st base (U) to G. P30-10-h1-3_del1 (SEQ ID NO: 28) deleted the 24th base 39-62 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 decreased (FIG. 12). Therefore, it was suggested that these mutated regions are important for binding to viral proteins. That is, the base sequence shown at the 12th to 24th positions from the 5'end of SEQ ID NO: 6 and the base sequence shown at the 39th to 62nd positions from the 5'end of SEQ ID NO: 6 may be required for binding to the viral protein. It is expected to be a core sequence (motif) or partially contain a core sequence.

Claims (17)

A / H1N1pdm09 influenza virus consisting of the base sequence represented by any of SEQ ID NOs: 4 to 11 or a nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the base sequence. On the other hand, a nucleic acid aptamer having a binding ability. A / H1N1pdm09 in the base sequence represented by any of SEQ ID NOs: 4 to 11 or in a secondary structure composed of a nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the base sequence. A nucleic acid aptamer having a binding ability to A / H1N1pdm09 influenza virus, which comprises a nucleic acid containing a base sequence shortened so as to retain the structure of a portion having a binding ability to the type influenza 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 fourth aspect of the present invention, wherein the chemical modification is a modification with a fluoro group (2'-F) or a methoxy group (2'-OMe) for the 2'position of the ribose moiety, or a substitution with hydrogen (2'-deoxy). Nucleic acid aptamer. The nucleic acid aptamer according to any one of claims 1 to 5, wherein the 5'end and / or the 3'end is modified. Single-stranded DNA, two, which contains the same base sequence as the nucleic acid aptamer according to claim 1 or 2 or a base sequence complementary to the base sequence and can be converted into the nucleic acid aptamer according to claim 1 or 2. Main strand DNA or RNA. A detection agent for A / H1N1pdm09 influenza virus containing the nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient. A diagnostic agent for A / H1N1pdm09 influenza virus containing the nucleic acid aptamer according to any one of claims 1 to 6 as an active ingredient. A method for detecting influenza A / H1N1pdm09, which comprises a step of allowing a nucleic acid aptamer according to any one of claims 1 to 6 to act on a sample to be inspected as an active ingredient. A test method for identifying or denying at least one of influenza virus subtypes, strains, and clades, which comprises the step of allowing a nucleic acid aptamer according to any one of claims 1 to 6 to act on a sample to be tested as an active ingredient. A nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
A nucleic acid aptamer in which the aptamer has a motif consisting of the base sequences represented by the 16th to 43rd positions from the 5'end of SEQ ID NO: 4. The motif forms at least one loop structure and
The nucleic acid aptamer according to claim 12, further comprising a stem structure formed by a base 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 or 13, which has 85% or more identity with the base sequence represented by the 1st to 58th positions from the 5'end of SEQ ID NO: 4. A nucleic acid aptamer capable of binding to A / H1N1pdm09 influenza virus.
The aptamer consists of the first motif consisting of the 12th to 24th base sequences from the 5'end of SEQ ID NO: 6 and the 39th to 62nd base sequences from the 5'end of SEQ ID NO: 6. Nucleic acid aptamer with a second motif. The aptamer has a loop structure formed by a base sequence containing the second motif, and is formed by bases added to the 5'end and 3'end of the base sequence constituting the loop structure, respectively. The nucleic acid aptamer of claim 15, further comprising a stem structure. The nucleic acid aptamer according to claim 15 or 16, which has 85% or more identity with respect to the base sequence represented by the 1st to 77th positions from the 5'end of SEQ ID NO: 6.

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