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WO2009123255A1 - Procédé et appareil de sélection d'un aptamère et procédé de détection utilisant l'aptamère - Google Patents

Procédé et appareil de sélection d'un aptamère et procédé de détection utilisant l'aptamère Download PDF

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Publication number
WO2009123255A1
WO2009123255A1 PCT/JP2009/056792 JP2009056792W WO2009123255A1 WO 2009123255 A1 WO2009123255 A1 WO 2009123255A1 JP 2009056792 W JP2009056792 W JP 2009056792W WO 2009123255 A1 WO2009123255 A1 WO 2009123255A1
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Prior art keywords
nucleic acid
stranded nucleic
double
binding substance
stranded
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Japanese (ja)
Inventor
稔 麻生川
巌 和賀
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NEC Corp
NEC Solution Innovators Ltd
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NEC Corp
NEC Solution Innovators Ltd
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Priority to JP2010505968A priority Critical patent/JP5317221B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the present invention relates to an aptamer selection method, a selection apparatus, a selection apparatus, and a sensor method and apparatus using the aptamer.
  • nucleic acids such as DNA, RNA and PNA have the property of binding specifically to specific molecules.
  • Such nucleic acids are generally called aptamers.
  • proteins there is a protein called an antibody, and the antibody has a property of specifically binding to a specific molecule (antigen).
  • Aptamers are similar to antibodies in that they specifically bind to a specific molecule, except that antibodies are proteins whereas aptamers are nucleic acids.
  • a target molecule a molecule to which an aptamer specifically binds.
  • the selected aptamer is not necessarily a nucleic acid having an optimal sequence for the target molecule. Therefore, in the SELEX method, in order to optimize the nucleic acid sequence selected in the above step as an aptamer, the selected nucleic acid is used as a template, amplified by Polymerase Chain Reaction (PCR), and based on the amplified product, Create a new nucleic acid pool.
  • PCR is one technique for amplifying a prepared nucleic acid as a template and making a large number of copies of the nucleic acid.
  • the nucleic acid amplified by PCR has the same sequence as the template nucleic acid sequence.
  • a part of the nucleic acid contained in the obtained amplification product can be a nucleic acid having a slightly different sequence from the template by performing amplification under conditions different from usual.
  • a nucleic acid pool is formed in which nucleic acids having the same sequence as the nucleic acid selected as the aptamer and nucleic acids having a variety of nucleic acid sequences slightly different from the sequence are mixed.
  • the obtained nucleic acid pool is poured into a column on which the target molecule is immobilized, and aptamers are selected again from the column.
  • nucleic acid sequence as an aptamer is optimized.
  • to amplify a nucleic acid means to prepare a large number of nucleic acid copies having the same sequence as the template based on the prepared amplification product using the prepared nucleic acid as a template, and using the prepared nucleic acid as a template.
  • nucleic acid of the same sequence of the template it refers to both production of a large amount of nucleic acid having a slightly different sequence from that nucleic acid.
  • Patent Document 2 (US 6706482B2), a method of performing the SELEX method without immobilizing a target molecule on a column as described in Patent Document 1 (US 5475096A1) is described.
  • a specific method one end of a nucleic acid to be selected is modified (labeled) with a compound such as a fluorescent dye, and a SELEX method is performed by using a column capable of selecting the compound (labeled).
  • Non-Patent Document 1 D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 (2007)
  • the target is as in Patent Document 1 (US 5475096A1).
  • a method of performing the SELEX method without immobilizing molecules on a column is described.
  • a nucleic acid fragment complementary to about 6 bases at the 5 'end of the aptamer candidate nucleic acid is used.
  • Biotin is bound to this nucleic acid fragment.
  • biotin is bound to the 5 'end of the aptamer candidate nucleic acid via a complementary nucleic acid fragment.
  • this aptamer candidate nucleic acid When this aptamer candidate nucleic acid is introduced into a column on which avidin is immobilized, the aptamer candidate nucleic acid is immobilized on the column due to the high affinity of biotin and avidin.
  • the target molecule is introduced into the column, if there is an aptamer having high binding ability with the target molecule, hybridization at the 5 ′ end of the aptamer candidate nucleic acid is broken, and aptamer having high binding ability is selected. can do.
  • Non-Patent Document 2 Manjula Rajendran et al., Nucleic Acids Res. Vol.31, no.19, pp.5700-5713. (2003)), it is targeted as in Patent Document 1 (US 5475096A1).
  • a method of performing the SELEX method without immobilizing molecules on a column is described. The specific method is very similar to the method disclosed in Non-Patent Document 1 (D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 (2007)).
  • a fluorescent quencher is bound to the end of the complementary nucleic acid fragment where biotin is not bound, and a fluorescent reagent is bound to the aptamer candidate nucleic acid.
  • the fluorescent reagent and the fluorescent quencher influence each other, and the fluorescent color is not developed.
  • Patent Document 1 (US 5475096A1) A method of performing the SELEX method without immobilizing the target molecule on the column is described. The specific method is similar to the method disclosed in Non-Patent Document 1 (DP Morse, Biochem. Biophys. Res. Commun., No.359, pp.94-101 (2007)). The complementary nucleic acid fragment hybridizes in the middle of the aptamer candidate nucleic acid.
  • the target molecule or aptamer candidate nucleic acid or a nucleic acid fragment complementary to the nucleic acid fragment must be modified (labeled). I must. It should be noted that hybridization between nucleic acids is not included in the modification treatment (labeling) referred to here.
  • Patent Document 1 US 5475096A1
  • modifying the target molecule is undesirable for the following reasons.
  • the target molecule since a part of the target molecule is changed by modification, there is a possibility that a site that can become an “epitope” (recognition part) of the aptamer disappears from the target molecule.
  • the target molecule since the target molecule is immobilized on a column or the like through a linker molecule, the display site from the target molecule to the aptamer is limited.
  • the target molecule is a low molecule, there is a problem that it is difficult to connect the target molecule and the linker molecule because there are few functional groups that can be used for the connection with the linker molecule.
  • immobilization of the target molecule not only prevents the aptamer from recognizing the target molecule, but also has a problem of low versatility because it cannot be technically used for all target molecules.
  • Patent Document 2 (US 6706482B2) and Non-Patent Documents 1 to 3 (D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 (2007), Manjula Rajendran et al., Nucleic Acids Res. Vol.31, no.19, pp.5700-5713. (2003), NutiuR, and Li Y., Angew. Chem. Int. Ed.
  • the aptamer candidate nucleic acid or a nucleic acid fragment complementary thereto is subjected to a treatment such as modification (labeling) without immobilizing the target molecule, and the aptamer candidate nucleic acid is immobilized on a column or the like.
  • Patent Document 2 In the method disclosed in Patent Document 2 (US Pat. No. 6,706,482 B2), one end of an aptamer candidate nucleic acid is modified (labeled) with a compound such as a fluorescent dye, and the nucleic acid is immobilized on a column via the compound. Therefore, in the method of Patent Document 2 (US 6706482 B2), one end of all aptamer candidates must be modified (labeled). This is very troublesome in terms of work process.
  • Non-Patent Documents 1-3 D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 (2007), Manjula Rajendran et al., Nucleic Acids Res. Vol. 31, no .19, pp.5700-5713. (2003), Nutiu R, and Li Y., Angew. Chem. Int. Ed. Engl, vol.44, pp.1061-1065, (2005))
  • a nucleic acid fragment complementary to a part of the aptamer candidate nucleic acid is used, and one end of the nucleic acid fragment is modified (labeled) and immobilized on the column.
  • the aptamer candidate nucleic acid is immobilized on the column through hybridization of nucleic acid fragments complementary to the aptamer candidate nucleic acid. Therefore, in the methods of Non-Patent Documents 1 to 3 (labeling), one end of all nucleic acid fragments complementary to aptamer candidates must be modified (labeled). This is very troublesome in terms of work process, as in Patent Document 2 (US Pat. No. 6,706,482 B2).
  • Patent Document 1 US 5475096A1
  • Patent Document 2 Patent Document 2
  • Non-Patent Documents 1 to 3 D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 ( 2007), Manjula Rajendran et al., Nucleic Acids Res. Vol. 31, no. 19, pp. 5700-5713. (2003), Nutiu R, and Li Y., Angew. Chem. Int. Ed. Engl, vol .44, pp.1061-1065, (2005)), there is low versatility as an aptamer selection method. In selecting aptamers, a wide variety of nucleic acid sequences are used.
  • Patent Document 1 US 5475096A1
  • Patent Document 2 US 6706482B2
  • Non-Patent Documents 1 to 3 D. P. Morse, Biochem. Biophys. Res. Commun., No. 359, pp. 94-101 ( 2007), Manjula Rajendran et al., Nucleic Acids Res. Vol. 31, no. 19, pp. 5700-5713. (2003), Nutiu R, and Li Y., Angew. Chem. Int. Ed. Engl, vol In the method of .44, pp.1061-1065, (2005)), each time an experiment is performed, the experimenter needs to perform the same modification treatment (labeling) on the target molecule or the aptamer candidate nucleic acid.
  • the aptamer selection method of the present invention comprises: A method for selecting an aptamer that is a nucleic acid that specifically binds to a molecule, Two nucleic acids formed by hybridization of at least part of the sequence of the first single-stranded nucleic acid that is an aptamer candidate with a double-stranded nucleic acid binding substance that specifically binds to the double-stranded portion of the nucleic acid A first step of binding to the chain portion; A second step of binding a first single-stranded nucleic acid and a molecule to which the first single-stranded nucleic acid specifically binds; And a third step in which the first single-stranded nucleic acid that binds to the molecule and the double-stranded nucleic acid-binding substance are separated from each other.
  • the aptamer selection device of the present invention is An aptamer selection device used for selecting an aptamer that is a nucleic acid that specifically binds to a molecule, A member, A double-stranded nucleic acid binding substance bound to the member, The double-stranded nucleic acid binding substance specifically binds to the double-stranded part of the nucleic acid formed by hybridizing at least part of the sequence of the single-stranded nucleic acid that is an aptamer candidate, and the single-stranded nucleic acid is specific.
  • the single-stranded nucleic acid and the double-stranded nucleic acid binding substance are separated from each other.
  • the sensor device of the present invention is A sensor device used to detect molecules, A member, A first double-stranded nucleic acid binding substance bound to the member; A double-stranded portion of a nucleic acid formed by hybridization of at least a portion of the sequence of a single-stranded nucleic acid having an aptamer of the molecule to which a first double-stranded nucleic acid-binding substance specifically binds. And When the molecule binds to the aptamer, the single-stranded nucleic acid and the first double-stranded nucleic acid binding substance are separated from each other.
  • the sensor device of the present invention comprises: A sensor device for detecting molecules, A member, A double-stranded nucleic acid binding substance bound to the member; A double-stranded portion of a nucleic acid formed by hybridization of at least a portion of a sequence of a single-stranded nucleic acid having an aptamer of a molecule, to which a double-stranded nucleic acid binding substance specifically binds; A nucleic acid detection means for detecting at least a part of the nucleic acid bound to the double-stranded nucleic acid binding substance and / or the nucleic acid separated from the double-stranded nucleic acid binding substance, When the molecule binds to the aptamer, the single-stranded nucleic acid and the double-stranded nucleic acid-binding substance are separated from each other.
  • the sensor device of the present invention comprises: A sensor device for detecting molecules, Members, A first double-stranded nucleic acid binding substance bound to the member; A double-stranded portion of a nucleic acid formed by hybridization of at least a portion of a sequence of a single-stranded nucleic acid having an aptamer of the molecule, specifically bound by a first double-stranded nucleic acid binding substance; A second double-stranded nucleic acid binding substance bound to the double-stranded portion of the nucleic acid; Double-stranded nucleic acid binding substance detection for detecting a second double-stranded nucleic acid binding substance bound to the double-stranded part of the nucleic acid and / or a second double-stranded nucleic acid binding substance separated from the double-stranded part of the nucleic acid Means, When the molecule binds to the aptamer, the single-stranded nucleic acid and the second double-stranded nucleic acid-
  • the sensor method of the present invention comprises: A sensor method for detecting molecules, Formed by hybridization of at least part of the sequence of a single-stranded nucleic acid having an aptamer of a molecule, a member, a double-stranded nucleic acid binding substance bound to the member, and a double-stranded nucleic acid binding substance specifically bound
  • the sensor method of the present invention comprises: A sensor method for detecting molecules, A member, a first double-stranded nucleic acid binding substance bound to the member, and a first double-stranded nucleic acid binding substance specifically bound to the first double-stranded nucleic acid binding substance, wherein at least part of the sequence of the single-stranded nucleic acid having an aptamer of the molecule is A double-stranded portion of a nucleic acid formed by hybridization and a second double-stranded nucleic acid binding substance bound to the double-stranded portion of the nucleic acid, and when the molecule binds to the aptamer, the single strand
  • a first step in which a molecule contacts an aptamer of a sensor device in which a nucleic acid and a second double-stranded nucleic acid-binding substance are separated from each other; After the first step, a second double-stranded nucleic acid binding substance bound to the double-stranded part of the nucle
  • a double-stranded nucleic acid binding substance that specifically binds to a double-stranded portion of a nucleic acid, it can be immobilized on a target molecule, aptamer candidate nucleic acid, or a nucleic acid fragment complementary to the nucleic acid fragment.
  • FIG. 1 is a diagram schematically showing a first embodiment of the present invention.
  • FIG. 2 is a diagram schematically showing a second embodiment of the present invention.
  • FIG. 3 is a diagram schematically showing a third embodiment of the present invention.
  • FIG. 4 is a diagram schematically showing a fourth embodiment of the present invention.
  • FIG. 5 is a diagram schematically showing a sixth embodiment of the present invention.
  • FIG. 6 is a diagram schematically showing a sixth embodiment of the present invention.
  • FIG. 7 is a diagram schematically showing a seventh embodiment of the present invention.
  • FIG. 8 is a diagram schematically showing a seventh embodiment of the present invention.
  • 1 to 8 have the following meanings.
  • the aptamer selection method and apparatus according to the present invention are such that at least a part of a single-stranded nucleic acid that is an aptamer candidate hybridizes with a nucleic acid fragment to form a double-stranded nucleic acid part.
  • An aptamer selection method and apparatus in which a double-stranded nucleic acid binding substance is bound to a member will be described.
  • the aptamer selection method and apparatus in this embodiment are shown in FIG.
  • the double-stranded nucleic acid binding substance 3 is added to the double-stranded part of the nucleic acid formed by hybridizing at least a part of the single-stranded nucleic acid 1 that is an aptamer candidate. Join.
  • the single-stranded nucleic acid 1 is hybridized with a nucleic acid fragment 2 which is a single-stranded nucleic acid having a sequence complementary to a part of the sequence to form a double-stranded nucleic acid portion (FIG. 1 (A)).
  • Double-stranded nucleic acid binding substance 3 binds to the double-stranded part of this nucleic acid (FIG. 1 (B)).
  • the double-stranded nucleic acid binding substance 3 has a property of specifically binding to the double-stranded part of the nucleic acid and not binding to the single-stranded part of the nucleic acid. Since the double-stranded nucleic acid binding substance 3 is bound to the member 4, the single-stranded acid 1 is also bound to the member 4 through the double-stranded nucleic acid binding substance 3.
  • the single-stranded nucleic acid 1 and the target molecule 5 are bound.
  • the double-stranded nucleic acid binding substance 3 is bound to the double-stranded nucleic acid portion of the single-stranded nucleic acid 1.
  • the target molecule 5 is brought close to the single-stranded nucleic acid 1 (FIG. 1C)
  • the target molecule 5 binds to the single-stranded nucleic acid 1.
  • FIG. 1D Conversely, when the single-stranded nucleic acid 1 is not an aptamer of the target molecule 5, the target molecule 5 does not bind to the single-stranded nucleic acid 1 (FIG. 1 (D)).
  • the single-stranded nucleic acid 1 that binds to the target molecule 5 and the double-stranded nucleic acid binding substance 3 are separated.
  • branch migration occurs, and the double-stranded portion of the nucleic acid disappears. This branch migration occurs when the binding between the target molecule 5 and the single-stranded nucleic acid 1 is stronger than the binding between the nucleic acids forming the double-stranded portion of the nucleic acid.
  • the double-stranded nucleic acid binding substance 3 cannot continue to bind to the single-stranded nucleic acid 1, and thus the single-stranded nucleic acid 1 and the double-stranded nucleic acid substance 3 are separated. (FIG. 1 (E)). Since the single-stranded nucleic acid 1 that is not an aptamer continues to have a double-stranded portion, it continues to bind to the double-stranded nucleic acid binding substance 3.
  • K D1 [single-stranded nucleic acid 1] ⁇ [nucleic acid fragment 2] / [single-stranded nucleic acid 1: nucleic acid fragment 2].
  • a hybrid of “(single-stranded nucleic acid 1 + target molecule 5)” and nucleic acid fragment 2 in which a single-stranded nucleic acid 1 and a target molecule 5 form a complex is a kind of complex.
  • dissociation constant K D2 [(single-stranded nucleic acid 1 + target molecule 5)] ⁇ [nucleic acid fragment 2] / [(single-stranded nucleic acid 1 + target molecule 5): nucleic acid fragment 2]
  • dissociation constant K D2 [(single-stranded nucleic acid 1 + target molecule 5)] ⁇ [nucleic acid fragment 2] / [(single-stranded nucleic acid 1 + target molecule 5): nucleic acid fragment 2]
  • the concentration of the single-stranded nucleic acid 1 and nucleic acid fragment 2 hybrid [single-stranded nucleic acid 1: nucleic acid fragment 2] also gradually decreases.
  • the dissociation constant KD2 of the hybrid (complex) of the complex of single-stranded nucleic acid 1 and molecule 5 (single-stranded nucleic acid 1 + molecule 5) and nucleic acid fragment 2 is different from the dissociation constant KD1. ing.
  • the dissociation constant K D2 is much larger than the dissociation constant K D1 and K D2 >> K D1 , the concentration of the target molecule 5 existing in the liquid phase [target molecule 5] is sufficiently high.
  • a complex of single-stranded nucleic acid 1 and target molecule 5 (single-stranded nucleic acid 1 + target molecule 5) accumulates in the liquid phase, and conversely, a hybrid of single-stranded nucleic acid 1 and nucleic acid fragment 2 And the double-stranded nucleic acid substance 3 and the complex disappear.
  • the above phenomenon occurs when the dissociation constant K D2 is significantly larger than the dissociation constant K D1 , and K D2 >> K D1 . This is particularly noticeable.
  • the single-stranded nucleic acid 1 that is not an aptamer continues to have a double-stranded portion. Continue to bind to substance 3.
  • the single-stranded nucleic acid 1 separated from the double-stranded nucleic acid binding substance 3 in the third step is recovered.
  • the single-stranded nucleic acid 1 that is an aptamer is separated from the double-stranded nucleic acid binding substance 3 and thus is also separated from the member 4. Therefore, for example, by flowing a solution or the like over the member 4, the single-stranded nucleic acid 1 is flowed simultaneously with the solution, and the single-stranded nucleic acid 1 that is an aptamer can be recovered.
  • the aptamer selection method according to the present invention makes it possible to select an aptamer without subjecting a target molecule, aptamer candidate nucleic acid, or a nucleic acid fragment complementary to the nucleic acid fragment to a modification treatment for immobilization. Therefore, there is no possibility that the epitope of the target molecule and the site to be presented to the aptamer disappear, and it is not necessary to fix the nucleic acid of the target molecule or aptamer candidate to the member.
  • the double-stranded nucleic acid binding substance 3 is bound to the member 4.
  • This aptamer selection apparatus is highly versatile because it can be applied to a wide variety of aptamer candidate nucleic acids and a wide variety of target molecules whose part of the sequence is involved in the formation of a double-stranded portion.
  • the aptamer selection apparatus according to the present invention can be reused any number of times as long as the binding ability to the double-stranded nucleic acid moiety of the double-stranded nucleic acid binding substance 3 is not lost.
  • the method for binding the double-stranded nucleic acid binding substance 3 to the member 4 may be a general immobilization method such as using a functional group, and the method is not particularly limited.
  • a functional group such as using a functional group
  • an amino group is added to the double-stranded nucleic acid binding substance 3 and a carboxyl group is added to the member 4, and the double-stranded nucleic acid binding substance 3 is immobilized on the member 4 by binding the amino group and the carboxyl group. Can do.
  • the double-stranded nucleic acid binding substance 3 used in the present invention represents a substance that specifically binds to the double-stranded part of the nucleic acid. Therefore, the double-stranded nucleic acid binding substance 3 does not bind to the single-stranded portion of the nucleic acid.
  • Examples of the double-stranded nucleic acid binding substance 3 include, but are not limited to, intercalators and nucleic acid binding proteins. Examples of intercalators include ethidium bromide, actinomycin, nogaramycin, distamycin A, and methidium. Examples of nucleic acid binding proteins include groove binders, zinc fingers, and leucine zippers.
  • Some of the double-stranded nucleic acid binding substances 3 specifically bind to a specific double-stranded sequence. By using many of these simultaneously, aptamer selection can be performed from a wide variety of aptamer candidates.
  • the nucleic acid used in the present invention may be DNA or RNA. Further, it may be a nucleic acid mimic such as PNA.
  • aptamer candidate single-stranded nucleic acids It is desirable to prepare a variety of aptamer candidate single-stranded nucleic acids. By preparing nucleic acids having sufficient variations, it is possible to select aptamers having higher binding ability.
  • the length of a typical aptamer sequence is 30 to 50 mer, but the length is not particularly limited. However, if the sequence is too short, the specificity as an aptamer is lowered. Conversely, when the nucleic acid is RNA, if the sequence is too long, it will be easily degraded by RNAase. It is desirable that the length of the nucleic acid is appropriately changed according to experimental conditions and experimental results.
  • a single-stranded nucleic acid that is an aptamer candidate is used as a template when preparing an amplification product to which a PCR method is applied, for example.
  • the obtained amplification product has a sequence complementary to the sequence of the downstream primer and the sequence corresponding to the sequence of the upstream primer, which is used for amplification using the PCR method. It will have at its 5 'end. Therefore, the amplification product obtained is a so-called “random sequence” portion having various sequences between the fixed region at the 3 ′ end and the 5 ′ end determined by the sequences of the downstream primer and the upstream primer. It has.
  • aptamer sequence corresponds to a region showing binding ability to a molecule according to the “random sequence” portion. That is, generally, the length of the “random sequence” portion is selected in the range of 30 mer to 50 mer, for example. On the other hand, the lengths of the sequences of the fixed regions at the 3 ′ end and the 5 ′ end depend on the sequences of the upstream primer and the downstream primer, which are appropriately selected according to the amplification conditions to which the PCR method is applied. Selected.
  • the promoter sequence of the RNA polymerase enzyme used for the in vitro transcription is provided at its 5 ′ end.
  • An upstream primer is used.
  • the 5 'end of the prepared single-stranded RNA has a sequence corresponding to the sequence of the upstream primer connected to the 3' end of the promoter sequence.
  • the double-stranded nucleic acid portion formed by the single-stranded nucleic acid that is an aptamer candidate may be in any portion of the single-stranded nucleic acid. It may be at the end of the single-stranded nucleic acid or in the middle. Further, the entire sequence of a single-stranded nucleic acid that is an aptamer candidate may be hybridized with a nucleic acid fragment to form a double-stranded part.
  • sequence of a single-stranded nucleic acid that is an aptamer candidate includes a so-called “random sequence” portion having various sequences between the 3′-end and 5′-end fixed regions as described above.
  • a configuration for forming a double-stranded portion with a nucleic acid fragment can be selected by using the above-mentioned fixed region at the 3 ′ end or 5 ′ end. In this case, it is possible to select an embodiment in which the entire sequence of the fixed region at the 3 'end or 5' end is hybridized with the nucleic acid fragment to form a double-stranded part.
  • the sequence of a part of the fixed region at the 3 'end or 5' end is hybridized with the nucleic acid fragment to form a double-stranded part.
  • the partial sequence can be selected at the 3 'end or 5' end of the single-stranded nucleic acid.
  • a structure in which a part of a so-called “random sequence” part is used to form a double-stranded part with a nucleic acid fragment is usually less frequently used.
  • the material of the member 4 is not particularly limited as long as the member 4 has no adsorptivity to nucleic acid or can be processed so as not to adsorb to nucleic acid.
  • the member 4 may be treated with a blocking agent or the like so that the nucleic acid is not adsorbed to the member 4.
  • the member 4 may be a substrate or a bead.
  • a conductive material is used for the member 4, it can also be used as the sensor method and apparatus according to the present invention described in the eighth embodiment.
  • the first to fourth steps are usually performed in a solution.
  • the solution to be used may be a general reaction solution used for complex formation reaction and complex dissociation reaction, and conditions such as temperature, pH, metal ion, etc. when performing each step are appropriately set.
  • Specific experimental conditions may be the same as the experimental conditions of the SELEX method.
  • Mg ions Mg 2+
  • the aptamer selection method of the present invention uses the double-stranded portion of the nucleic acid, temperature conditions, pH, and the like that cannot maintain the double-stranded bond of the nucleic acid are undesirable. Also, a condition that the ability to specifically bind to the double-stranded nucleic acid of the double-stranded nucleic acid binding substance 3 is lost is not desirable.
  • the binding force between the first single-stranded nucleic acid 1 and the nucleic acid fragment 2 is strong, even when the target molecule 5 touches the first single-stranded nucleic acid 1 that is an aptamer, the aptamer and the target molecule 5 do not bind, The aptamer remains immobilized on the member 4 via the double-stranded nucleic acid binding substance 3.
  • experimental conditions such as temperature and pH. For example, by setting the temperature to the melting temperature (Tm) of the double-stranded portion of the nucleic acid, the double-stranded portion of the nucleic acid can be easily dissolved.
  • the melting temperature (Tm) of the double-stranded part of the nucleic acid is a temperature at which the double-stranded nucleic acid becomes a single-stranded nucleic acid by heat denaturation.
  • Tm melting temperature
  • a primer to the single-stranded nucleic acid used as the aptamer candidate.
  • it is desirable to add a primer when optimizing the nucleic acid sequence of the selected aptamer, it is desirable to add a primer.
  • a primer is added only to a single-stranded nucleic acid that is an aptamer candidate without adding a primer to the nucleic acid fragment, the nucleic acid fragment recovered together with the aptamer is not amplified, thus improving the performance of aptamer selection. be able to.
  • the aptamer selection method When applying the aptamer selection method according to the present invention, it is possible to increase the specificity for the target molecule depending on the device. For example, by the method shown below, it is possible to extract an aptamer that does not bind to a compound having a structure similar to the target molecule but binds only to the target molecule.
  • a compound having a structure similar to the target molecule is brought into contact with the nucleic acid. Then, the nucleic acid that binds to the compound having a structure similar to the target molecule and is separated from the double-stranded nucleic acid binding substance is removed. Then, the second and subsequent steps are performed.
  • the specificity of the aptamer to the target molecule can be increased.
  • the aptamer selection method and apparatus according to the present invention are such that a part of a single-stranded nucleic acid that is an aptamer candidate hybridizes with a nucleic acid fragment to form a double-stranded nucleic acid part.
  • the aptamer selection method according to the present invention does not require a modification treatment for immobilization of a target molecule, a single-stranded nucleic acid of an aptamer candidate, or a nucleic acid fragment complementary to the single-stranded nucleic acid.
  • the aptamer selection apparatus is highly versatile and can be used for single-stranded nucleic acids of various aptamer candidates and target molecules.
  • the aptamer selection device can be reused as long as the binding ability to the double-stranded nucleic acid possessed by the double-stranded nucleic acid binding substance is not lost. According to the present invention, it is possible to improve the simplicity, efficiency, and productivity of aptamer selection operation, and to realize an apparatus with high versatility and reusability.
  • the above-mentioned “random sequence” portion in the single-stranded nucleic acid 1 is selected as the aptamer candidate.
  • a “random sequence” having a length of 30 mer to 50 mer is prepared.
  • At least a part of the single-stranded nucleic acid 1 hybridizes with the nucleic acid fragment 2 to form a double-stranded nucleic acid portion, and the double-stranded nucleic acid binding substance 3 bound to the member 4 causes the double-stranded nucleic acid to bind to the double-stranded nucleic acid.
  • Examples of such a double-stranded nucleic acid binding substance 3 include, but are not limited to, an intercalator and a nucleic acid binding protein.
  • Examples of the intercalator include nitrogen-containing condensed ring compounds such as acridine and ethidium bromide, methylene blue, benzopyrene, actinomycin, nogaramycin, distamycin A, methidium, and derivatives thereof.
  • This intercalator includes a linker that is used as the member 4 and can be fixed to the support.
  • a resonance plasmon measuring device for example, Biacore, etc.
  • a gold substrate for the sensor chip of the resonance plasmon measuring apparatus is used as the body.
  • a phosphate buffer solution in which sodium chloride is dissolved can be used as the reaction solution, but is not limited thereto.
  • an aqueous solution of thrombin is added so that the thrombin of the target molecule 5 has a predetermined concentration.
  • the sensor chip is incubated at room temperature for 2 hours in a reaction solution containing the predetermined concentration of thrombin.
  • the aptamer selection method according to the present invention is such that a single-stranded nucleic acid 6 that is an aptamer candidate hybridizes with the sequence of the single-stranded nucleic acid itself to form a double-stranded nucleic acid portion.
  • the aptamer selection method is described.
  • FIG. 2 shows an aptamer selection method and apparatus in the second embodiment. Since the second embodiment is an application of the first embodiment, the description of the same points as the first embodiment will be omitted.
  • the single-stranded nucleic acid 6 of the aptamer candidate used in the second embodiment at least a part of the sequence of the single-stranded nucleic acid 6 and at least a part of the sequence of the single-stranded nucleic acid 6 are hybridized to form a nucleic acid. Are formed (FIG. 2 (A)).
  • the first, second, third, and fourth steps may be performed in the same manner as in the first embodiment.
  • the apparatus used for the aptamer selection method may be the same apparatus as that used in the first embodiment.
  • the double-stranded part of the nucleic acid formed by the single-stranded nucleic acid 6 has a hairpin structure, a pseudoknot structure, etc., but it is sufficient that it forms a double-stranded part, and the structure is not limited to these. Absent.
  • dissociation of the complex “(tertiary structure + target molecule 5)” of the tertiary structure of the single-stranded nucleic acid 6 and the target molecule 5 proceeds according to the dissociation constant KD2 .
  • the concentration [target molecule 5] of the target molecule 5 present in the liquid phase is sufficiently high, the released tertiary structure of the single-stranded nucleic acid 6 is complexed with the target molecule 5 in the liquid phase. Form the body quickly.
  • Complex “(tertiary structure + target molecule 5)” of structure and target molecule 5 and complex “(tertiary structure + target molecule 5): double-stranded nucleic acid binding substance 3” Is different from the dissociation constant KD3 .
  • the concentration of the target molecule 5 present in the liquid phase [target molecule 5] is sufficiently high.
  • a complex of the tertiary structure of the single-stranded nucleic acid 6 and the target molecule 5 (tertiary structure + target molecule 5) accumulates.
  • a very small part is a complex “(tertiary structure) of a tertiary structure of a single-stranded nucleic acid 6 and a target molecule 5“ (tertiary structure + target molecule 5) ”and a double-stranded nucleic acid binding substance 3.
  • Target molecule 5 constituting double-stranded nucleic acid binding substance 3 ”, but almost all of the complex of tertiary structure of single-stranded nucleic acid 6 and target molecule 5 (tertiary structure + target molecule 5) is liquid Accumulate during the phase. Eventually, a complex of the tertiary structure of the single-stranded nucleic acid 6 and the target molecule 5 (tertiary structure + target molecule 5) accumulates in the liquid phase, and conversely, the tertiary structure of the single-stranded nucleic acid 6 The body, the double-stranded nucleic acid substance 3 and most of the complex disappear.
  • the dissociation constant K D3 is much larger than the dissociation constant K D2 , and K D3 >> K D2 In some cases, it progresses particularly noticeably.
  • the aptamer selection method according to the present invention in which a single-stranded nucleic acid that is an aptamer candidate hybridizes with its own sequence to form a double-stranded nucleic acid portion, has been described.
  • a single-stranded nucleic acid hybridizes with its own sequence to form a double-stranded nucleic acid moiety, another nucleic acid fragment having a sequence complementary to the single-stranded nucleic acid is not necessary.
  • the aptamer selection method according to the present invention is an aptamer selection method for binding a nucleic acid and a target molecule before binding a double-stranded portion of a nucleic acid that is an aptamer candidate to a double-stranded nucleic acid binding substance.
  • a method will be described.
  • the aptamer selection method in the third embodiment is shown in FIG. Since the third embodiment is an application of the first embodiment, the description of the same points as the first embodiment and the second embodiment will be omitted.
  • a single-stranded nucleic acid 1 having a double-stranded part formed by hybridizing at least a part of the sequence is prepared as an aptamer candidate (FIG. 3A). This binds to the target molecule 5 (FIG. 3B). At this time, only the single-stranded nucleic acid 1 that is an aptamer leaves the nucleic acid fragment 2 and binds to the target molecule 5. The single-stranded nucleic acid 1 that is not an aptamer continues to have a double-stranded part while being hybridized with the nucleic acid fragment 2.
  • the second step of the third embodiment in order to separate the substance formed by the first step and the nucleic acid having a double-stranded portion and the substance in which the target molecule 5 and the single-stranded nucleic acid 1 are bound, Only the nucleic acid having a strand portion is bound to the double-stranded nucleic acid binding substance 3 (FIGS. 3C and 3D). Since the substance in which the target molecule 5 and the single-stranded nucleic acid 1 are bound does not have the double-stranded portion of the nucleic acid, it does not bind to the double-stranded nucleic acid binding substance 3.
  • the double-stranded portion of the nucleic acid having a double-stranded portion binds to the double-stranded nucleic acid binding substance 3.
  • the nucleic acid contained in the double-stranded part is immobilized on the member via the double-stranded nucleic acid binding substance 3.
  • the single-stranded nucleic acid 1 that is an aptamer is recovered.
  • a double-stranded portion that is not an aptamer is poured by pouring a solution containing a substance in which the target molecule 5 and the single-stranded nucleic acid 1 are bonded and a nucleic acid having a double-stranded portion onto the substrate. It becomes possible to recover only the single-stranded nucleic acid 1 which is an aptamer by binding only the nucleic acid having a chain to the substrate.
  • the single-stranded nucleic acid 1 that is an aptamer candidate may be one that hybridizes with the nucleic acid fragment 2 to form a double-stranded nucleic acid, or doubles with the sequence of the single-stranded nucleic acid 1 itself. A part may be formed.
  • the aptamer selection method according to the present invention is a method for binding a single-stranded nucleic acid and a target molecule before binding a double-stranded portion of a nucleic acid that is an aptamer candidate to a double-stranded nucleic acid binding substance.
  • the aptamer selection method to be performed was described.
  • the aptamer candidate nucleic acid having a double-stranded portion may be bound to either the double-stranded nucleic acid binding substance or the target molecule first.
  • an aptamer selection method according to the present invention which is an aptamer selection method using a double-stranded nucleic acid binding substance having a tag, will be described.
  • the aptamer selection method in the fourth embodiment is shown in FIG. Since the fourth embodiment is an application of the first to third embodiments, the description of the same points as the first to third embodiments will be omitted.
  • a nucleic acid formed by hybridizing at least a part of the sequence of the single-stranded nucleic acid 1 that is an aptamer candidate The double-stranded part of (2) binds to the double-stranded nucleic acid binding substance 3 (FIG. 4B).
  • the double-stranded nucleic acid binding substance 3 is not bound to the member 4 in advance (FIG. 4A). Since the double-stranded nucleic acid binding substance 3 is not bound to the member 4, the single-stranded nucleic acid 1 bound to the double-stranded nucleic acid binding substance 3 is not immobilized on the member.
  • the single-stranded nucleic acid 1 and the target molecule 5 are bound (FIG. 4C).
  • the single-stranded nucleic acid 1 is an aptamer, it binds to the target molecule 5, but when the single-stranded nucleic acid 1 is not an aptamer, the target molecule 5 does not bind to the single-stranded nucleic acid 1 ( FIG. 4 (D)).
  • the single-stranded nucleic acid 1 that binds to the target molecule 5 and the double-stranded nucleic acid binding substance 3 are separated (FIG. 4 (E)).
  • the target molecule 5 binds to the single-stranded nucleic acid 1 that is an aptamer in the second step
  • the double-stranded portion of the nucleic acid disappears, so the double-stranded nucleic acid binding substance 3 cannot bind to the single-stranded nucleic acid 1
  • the single-stranded nucleic acid 1 and the double-stranded nucleic acid material 3 are separated. Since the single-stranded nucleic acid 1 that is not an aptamer continues to have a double-stranded portion, it continues to bind to the double-stranded nucleic acid binding substance 3.
  • the single-stranded nucleic acid 1 separated from the double-stranded nucleic acid binding substance 3 in the third step is recovered (FIG. 4 (F), FIG. 4 (G)).
  • the nucleic acid bound to the double-stranded nucleic acid binding substance 3 is not an aptamer, it is not desirable to recover the nucleic acid bound to the double-stranded nucleic acid binding substance 3.
  • the fourth step is realized by selectively separating the double-stranded nucleic acid binding substance 3.
  • the double-stranded nucleic acid binding substance 3 of this embodiment has a tag.
  • the tag that the double-stranded nucleic acid binding substance 3 has indicates a structure or property that allows the double-stranded nucleic acid binding substance 3 to be selectively extracted, and examples thereof include His-tag protein and GST fusion protein. However, it is not limited to these.
  • the member 4 in the fourth embodiment is a substance having a high affinity with the tag of the double-stranded nucleic acid binding substance 3, and the member 4 varies depending on the tag to be used. For example, when using a His-tag protein, cobalt resin or the like having a high affinity with the His-tag protein is desirable, and when using a GST fusion protein, glutathione beads or the like are desirable.
  • the single-stranded nucleic acid 1 that is an aptamer candidate may be one that hybridizes with the nucleic acid fragment 2 to form a double-stranded nucleic acid moiety, or two that hybridize with the sequence of the single-stranded nucleic acid 1 itself.
  • a chain part may be formed.
  • the aptamer selection method according to the present invention which is a method of aptamer selection using a double-stranded nucleic acid binding substance having a tag, has been described. If it is possible to selectively extract a double-stranded nucleic acid binding substance by attaching a tag to the double-stranded nucleic acid binding substance, aptamer selection is performed without previously binding the double-stranded nucleic acid substance to a member. Can do.
  • the aptamer selection method according to the present invention is the fifth step in which the single-stranded nucleic acid recovered in the fourth step is amplified, and the single-stranded nucleic acid amplified in the fifth step is the first step.
  • the first single-stranded nucleic acid of step 1 to determine the base sequence of the single-stranded nucleic acid that is the aptamer
  • the sixth step of repeating the first to fifth steps as necessary A method having the seventh step will be described. Since the fifth embodiment is an application of the first to fourth embodiments, the description of the same points as in the first to fourth embodiments will be omitted.
  • the single-stranded nucleic acid that is an aptamer is recovered by the first to fourth steps described in the first to fourth embodiments.
  • the single-stranded nucleic acid is recovered.
  • a template using Polymerase Chain Reaction (PCR) or the like as a template.
  • the single-stranded nucleic acid amplified in the fifth step is used as the first single-stranded nucleic acid in the first step, and if necessary, the first step Repeat the fifth step.
  • aptamers can be selected from the library of single-stranded nucleic acids prepared first.
  • the aptamer selected here is not necessarily the optimum aptamer for the target molecule. Therefore, by repeating the first to fifth steps and optimizing the sequence of the single-stranded nucleic acid that is an aptamer, it is possible to select an aptamer with higher affinity with the target molecule.
  • the single-stranded nucleic acid is amplified so that a part of the sequence of the single-stranded nucleic acid is mutated.
  • many kinds of single-stranded nucleic acids having a slightly different sequence from the sequence are generated. .
  • the first to fifth steps are repeated to optimize the sequence as an aptamer. To do.
  • the base sequence of a single-stranded nucleic acid that is an aptamer selected by the aptamer selection method of the present invention is determined.
  • the base sequence can be determined by a commonly used sequencing method, and the method is not particularly limited.
  • the obtained aptamer may be amplified by PCR and directly sequenced, or may be cloned after cloning using a vector.
  • the aptamer when the first step to the fifth step are repeated a plurality of times, the aptamer is initially subjected to conditions under which the aptamer is likely to bind to the target molecule or where the double-stranded portion of the aptamer candidate nucleic acid is fragile. It is desirable to make these conditions stricter as selections are made and repeated. If aptamer selection is performed from the beginning under conditions where it is difficult for the aptamer to bind to the target molecule or under conditions where the double-stranded portion of the aptamer is difficult to break, the aptamer may not be recovered in the fourth step.
  • the conditions for aptamer selection are made stricter. Optimize sequences as aptamers. And aptamers with high affinity can be selected.
  • the conditions under which an aptamer is likely to bind to a target molecule are conditions under which the binding force between the aptamer and the target molecule becomes strong, and the conditions can be set by changing experimental conditions such as temperature and pH.
  • the dissociation constant K D [aptamer] ⁇ [target molecule] / [target molecule / (target molecule) complex “(aptamer + target molecule)”. (Aptamer + target molecule)] corresponds to a small condition.
  • the dissociation constant, K D is generally formed of the complex energy Delta] E (where, Delta] E ⁇ 0) with respect to the temperature T, exhibits the dependence of exp ( ⁇ E / kT), the temperature T, and, pH It changes by changing ⁇ E depending on solution conditions such as.
  • the aptamer forms a tertiary structure.
  • the equilibrium constant K 1 also generally shows the dependence of exp ( ⁇ E 1 / kT) on the formation energy ⁇ E 1 (where ⁇ E 1 ⁇ 0) and the temperature T of the tertiary structure, the temperature T, This is changed by changing ⁇ E 1 depending on the solution conditions such as pH.
  • the conditions under which the double-stranded portion of the aptamer candidate nucleic acid is fragile are conditions under which the binding force between the nucleic acids weakens, and the conditions can be set by changing experimental conditions such as temperature and pH.
  • one of the conditions where the double-stranded portion of the aptamer candidate nucleic acid is fragile is a single-stranded nucleic acid / nucleic acid fragment hybrid “single-stranded nucleic acid: nucleic acid fragment”
  • the dissociation constant K D1 [single stranded nucleic acid] ⁇ [nucleic acid fragment] / [single stranded nucleic acid: nucleic acid fragment].
  • the dissociation constant K D1 also generally shows the dependence of exp ( ⁇ E H / kT) on the formation energy ⁇ E H (where ⁇ E H ⁇ 0) and the temperature T of the hybrid, so that the temperature T, It changes by changing ⁇ E H depending on the solution conditions such as pH.
  • the fifth step in order to amplify the single-stranded nucleic acid of the aptamer by PCR, it is desirable to previously add a primer to the single-stranded nucleic acid used for selection. By attaching the primer, amplification by PCR can be easily performed.
  • the single-stranded nucleic acid prepared in the first step is hybridized with the nucleic acid fragment to form a double-stranded nucleic acid portion, the single-stranded nucleic acid that is an aptamer in the fourth step and the non-aptamer nucleic acid fragment Both will be recovered.
  • a primer is attached only to the single-stranded nucleic acid that is an aptamer, only the aptamer can be amplified in the fifth step.
  • the mutation given to the single-stranded nucleic acid can be caused by increasing the error rate of reverse transcriptase or polymerase by manipulating the experimental conditions when performing reverse transcription or PCR.
  • the experimental conditions include the amount and quality of aptamer sequences used as templates, the amount of Mg and dNTP added to the reaction solution, PCR efficiency, the presence of inhibitors, and the number of cycles.
  • the seventh step when determining the sequence of the single-stranded nucleic acid that is an aptamer, it is desirable to produce a large number of copies using the single-stranded nucleic acid as a template by PCR performed in the fifth step.
  • the single-stranded nucleic acid is amplified so that no mutation occurs in the single-stranded nucleic acid to be produced. Making large copies of single-stranded nucleic acids makes it easier to determine the sequence.
  • the aptamer selection method is the fifth step of amplifying the single-stranded nucleic acid collected in the fourth step, and the single-stranded nucleic acid amplified in the fifth step.
  • a sixth step of using the first single-stranded nucleic acid of the first step and repeating the first to fifth steps as necessary, and a seventh step of determining the base sequence of the single-stranded nucleic acid that is an aptamer The method of having been described. By carrying out these steps, it is possible to optimize the aptamer sequence and to select aptamers having high specificity for the target molecule. It is also possible to determine the sequence of a single-stranded nucleic acid selected as an aptamer.
  • 6th Embodiment it is the apparatus for sensors used in order to detect the molecule
  • a sensor device and method using an aptamer according to the sixth embodiment will be described with reference to FIGS. Note that the description of the same points as in the sixth embodiment and the first to fifth embodiments will be omitted.
  • FIG. 5 (A) and FIG. 6 (A) show a sensor device 10 using an aptamer according to the present invention.
  • a double-stranded nucleic acid binding substance 3 is bound to the member 4.
  • An aptamer single-stranded nucleic acid 1 is hybridized with a nucleic acid fragment 2 having a sequence complementary to that sequence to form a double-stranded portion of the nucleic acid.
  • the single-stranded nucleic acid 1 that is an aptamer is bound to the member 4 through this double-stranded part.
  • a label 7 is added to each of the single-stranded nucleic acid 1 and the nucleic acid fragment 2.
  • the single-stranded nucleic acid 1 that is an aptamer may be a nucleic acid having an aptamer sequence that specifically binds to a target molecule.
  • the single-stranded nucleic acid 1 of the aptamer included in the sensor device 10 and the test object 11 are in contact with each other (FIGS. 5B and 6B).
  • the target molecule 5 and the single-stranded nucleic acid 1 that is an aptamer bind to each other.
  • the double-stranded nucleic acid portion of the single-stranded nucleic acid 1 is released by branch migration, and thus the double-stranded portion of the nucleic acid is eliminated.
  • the single-stranded nucleic acid 1 is separated from the double-stranded nucleic acid binding substance 3 (FIG. 5C).
  • the molecule 8 that is not the target molecule contained in the test object 11 does not bind to the single-stranded nucleic acid 1 that is an aptamer. Therefore, the single-stranded nucleic acid 1 continues to be bound to the double-stranded nucleic acid binding substance 3 (FIG. 6C).
  • nucleic acid separated from the double-stranded nucleic acid binding substance 3 is detected.
  • the target molecule 5 is included in the test object 11 used in the first step
  • the single-stranded nucleic acid 1 and the nucleic acid fragment 2 that are aptamers are separated from the double-stranded nucleic acid binding substance 3. If the nucleic acid separated from the double-stranded nucleic acid binding substance 3 can be detected, it can be determined that the target molecule 5 is included in the test object 11. On the contrary, if the nucleic acid separated from the double-stranded nucleic acid binding substance 3 cannot be detected, it can be determined that the target molecule 5 is not included in the test object 11.
  • the sixth embodiment it is possible to confirm whether the target molecule 5 is included in the test object 11 by detecting the nucleic acid that binds to the double-stranded nucleic acid binding substance 3 in the second step. .
  • the amount of nucleic acid bound to the double-stranded nucleic acid binding substance 3 is examined and compared with the amount of nucleic acid bound to the double-stranded nucleic acid binding substance 3 before the first step. To do. If the amount of nucleic acid bound to the double-stranded nucleic acid binding substance 3 after the first step is less than that before the first step, the target molecule 5 is included in the test object, and thus the aptamer. It can be determined that the single-stranded nucleic acid 1 is separated from the double-stranded nucleic acid binding substance 3.
  • the aptamer binding device can be used for versatility and reusability. high.
  • a device for detecting nucleic acid separated from the double-stranded nucleic acid binding substance 3 or a means for detecting at least a part of the nucleic acid binding to the double-stranded nucleic acid binding substance 3 is added to the apparatus shown in FIG. This device becomes a sensor device.
  • the nucleic acid for example, at least a part of the nucleic acid may be labeled.
  • the single-stranded nucleic acid 1 that is an aptamer or the nucleic acid fragment 2 complementary to the aptamer is previously labeled 7 with a fluorescent dye, a radioisotope (RI), a fluorescent quencher, a fluorescent reagent, etc.
  • RI radioisotope
  • the amount of nucleic acid can be detected.
  • the label 7 is given to the single-stranded nucleic acid 1 and the nucleic acid fragment 2, but the label 7 may be given to only one of them.
  • nucleic acids separated from the double-stranded nucleic acid binding substance 3 it is desirable to remove the nucleic acids separated from the double-stranded nucleic acid binding substance 3 from the member 4 after the first step and collect the nucleic acids. .
  • the amount (concentration) of the nucleic acid used for detection can be increased, and the detection sensitivity is increased.
  • nucleic acid In order to detect nucleic acid, it is not always necessary to label in advance. For example, it is possible to determine whether or not there is a nucleic acid separated from the double-stranded nucleic acid binding substance 3 by examining the nucleic acid contained in the collected material using a spectrophotometer or a mass spectrometer.
  • the nucleic acid binding to the double-stranded nucleic acid binding substance 3 and checking whether or not the target molecule 5 is contained in the object to be inspected 11 it is separated from the double-stranded nucleic acid binding substance 3 after the first step. It is desirable to remove the nucleic acid from the member 4. By removing, the amount of nucleic acid that binds to the double-stranded nucleic acid binding substance 3 after the first step can be accurately measured. In addition, the amount of nucleic acid bound to the double-stranded nucleic acid binding substance 3 after the first step can be accurately compared with the amount of nucleic acid bound to the double-stranded nucleic acid binding substance 3 before the first step. .
  • the amount of nucleic acid that binds to the double-stranded nucleic acid binding substance 3 before the first step is measured by measuring the signal intensity derived from the label 7 possessed by the nucleic acid that binds to the double-stranded nucleic acid binding substance 3 before the first step. Can be investigated.
  • the amount of the nucleic acid binding to the double-stranded nucleic acid binding substance 3 before the first step is known in advance, the label 7 included in the nucleic acid binding to the double-stranded nucleic acid binding substance 3 before the first step is used. There is no need to measure the derived signal strength.
  • the nucleic acid is detected by using a fluorescent quencher and a fluorescent reagent. If no signal is emitted when the single-stranded nucleic acid moiety is formed and no signal is emitted when the nucleic acid is a single-stranded nucleic acid, the nucleic acid removal step is not necessary.
  • the aptamer selected in the first to fifth embodiments is desirable to use as the aptamer. This is because the double-stranded nucleic acid moiety formed by the aptamer selected in the first to fifth embodiments binds to the double-stranded nucleic acid binding substance 3. In addition, when the target molecule is bound to the aptamer, the double-stranded portion of the nucleic acid is eventually unwound and finally the aptamer is separated from the double-stranded nucleic acid binding substance 3.
  • the single-stranded nucleic acid 1 that is an aptamer candidate may be one that hybridizes with the nucleic acid fragment 2 to form a double-stranded nucleic acid, or doubles with the sequence of the single-stranded nucleic acid 1 itself. A part may be formed.
  • the sensor device, the sensor device, and the method using the aptamer according to the present invention which can confirm the presence of the target molecule by detecting the nucleic acid, have been described.
  • the sensor device in the sixth embodiment can be said to be excellent in versatility and reusability since the devices used in the first to fifth embodiments can be used as they are.
  • the aptamer selected in the first to fifth embodiments is used, the aptamer can be used as it is without confirming that the aptamer is separated from the double-stranded binding substance when the target molecule is bound to the aptamer. Can be used.
  • the sensor device, sensor device and method of the present invention can be applied to various sensors.
  • narcotics such as cocaine and explosives selected using the first to fifth embodiments.
  • an aptamer for a dangerous substance it can be used as a sensor device and a method for detecting a dangerous substance such as a narcotic drug such as cocaine or an explosive as a target molecule.
  • the first double-stranded nucleic acid binding substance 3 is bound to the member 4.
  • An aptamer single-stranded nucleic acid 1 is hybridized with a nucleic acid fragment 2 having a sequence complementary to that sequence to form a double-stranded portion.
  • the first double-stranded nucleic acid binding substance 3 and the second double-stranded nucleic acid binding substance 9 not bound to the member 4 are bound to the double-stranded part.
  • the second double-stranded nucleic acid binding substance 9 is immobilized on the member 4 via the double-stranded portion of the nucleic acid and the first double-stranded nucleic acid binding substance 3.
  • the single-stranded nucleic acid 1 of the aptamer included in the sensor device 10 and the test object 11 are in contact with each other (FIGS. 7B and 8B).
  • the target molecule 5 and the single-stranded nucleic acid 1 that is an aptamer bind to each other.
  • the double-stranded nucleic acid portion of the single-stranded nucleic acid 1 is released by branch migration, and thus the double-stranded portion of the nucleic acid is eliminated.
  • the single-stranded nucleic acid 1 is separated from the first double-stranded nucleic acid binding substance 3.
  • the second double-stranded nucleic acid binding substance 9 is also separated from the single-stranded nucleic acid 1 and the first double-stranded nucleic acid binding substance 3 (FIG. 7C).
  • the substance 8 that is not the target molecule contained in the test object 11 does not bind to the single-stranded nucleic acid 1 that is an aptamer.
  • the second double-stranded nucleic acid binding substance 9 continues to bind to the double-stranded portion of the nucleic acid (FIG. 8C).
  • the second double-stranded nucleic acid binding substance 9 separated from the single-stranded nucleic acid 1 is detected.
  • the target molecule 5 is included in the test object 11 used in the first step
  • the second double-stranded nucleic acid binding substance 9 is separated from the single-stranded nucleic acid 1. If the second double-stranded nucleic acid binding substance 9 separated from the single-stranded nucleic acid 1 can be detected, it can be determined that the target molecule 5 is contained in the test object 11. Conversely, if the second double-stranded nucleic acid binding substance 9 that is separated from the single-stranded nucleic acid 1 cannot be detected, it can be determined that the target molecule 5 is not included in the test object 11.
  • the target molecule 5 is included in the test object 11 by detecting the second double-stranded nucleic acid binding substance 9 that binds to the single-stranded nucleic acid 1 in the second step. It can also be confirmed. After the first step, the amount of the second double-stranded nucleic acid binding substance 9 bound to the single-stranded nucleic acid 1 was examined, and the amount was bound to the single-stranded nucleic acid 1 before the first step. The amount of the second double-stranded nucleic acid binding substance 9 is compared.
  • the target molecule 5 is present on the test object 11. It can be determined that it was included.
  • the sensor device according to the present invention can use the aptamer selection device used in the first to fifth embodiments or the aptamer selection device combined with the aptamer selection device, it is versatile and reusable. It can be said that it is excellent.
  • the second double-stranded nucleic acid binding substance 9 may be labeled.
  • the second double-stranded nucleic acid binding substance 9 By labeling the second double-stranded nucleic acid binding substance 9 in advance with a fluorescent dye, radioisotope (RI), fluorescent quencher, etc., and measuring the signal intensity derived from these labels, the second two The amount of the strand nucleic acid binding substance 9 can be detected.
  • the second double-stranded nucleic acid binding substance 9 has a tag, the second double-stranded nucleic acid binding substance 9 separated from the single-stranded nucleic acid 1 is recovered, and the second double-stranded nucleic acid is recovered.
  • the amount of the second double-stranded nucleic acid binding substance 9 immobilized on the member 4 can be examined by measuring the current flowing through the member 4. In this case, it is desirable that the first double-stranded nucleic acid binding substance 3, the second double-stranded nucleic acid binding substance 9, and the member 4 are conductive. This method will be described below.
  • the current flowing through the member 4 (FIGS. 7A and 8A) before the test object 11 is brought into contact with the single-stranded nucleic acid 1 of the aptamer is measured.
  • the current flows to the member 4, the first double-stranded nucleic acid binding substance 3 bound to the member 4, and the second double-stranded nucleic acid binding substance 9 immobilized on the member 4 through the double-stranded portion of the nucleic acid. .
  • the current flowing through the member 4 after the inspection object 11 is brought into contact with the aptamer is measured (FIGS. 7C and 8C).
  • the target molecule 5 that binds to the aptamer is included in the test object 11
  • the double-stranded part of the nucleic acid disappears as a result.
  • the double-stranded nucleic acid binding substance 9 is separated from the double-stranded portion of the nucleic acid and also away from the member 4 (FIG. 7C). Therefore, the magnitude of the current flowing through the member 4 is smaller after the object to be inspected 11 is brought into contact with the aptamer than before being brought into contact.
  • the second double-stranded nucleic acid binding substance 9 continues to be immobilized on the member 4 through binding to the double-stranded portion of the nucleic acid.
  • the magnitude of the current flowing through the member 4 does not change.
  • the presence or absence of the target molecule 5 can be examined by comparing the magnitude of the current flowing through the member 4 before and after the test object 11 is brought into contact with the aptamer.
  • a means for detecting the second double-stranded nucleic acid binding substance 9 separated from the single-stranded nucleic acid 1 or a second double-stranded nucleic acid that binds to the single-stranded nucleic acid 1 Means for detecting the binding substance 9, or means for passing a current through the member 4, the first double-stranded nucleic acid binding substance 3 bound to the member 4 and the second double-stranded nucleic acid binding substance 9, and the current If a means for measuring is added, this device becomes a sensor device.
  • the number of the second double-stranded nucleic acid binding substance 9 that binds to the single-stranded nucleic acid 1 that is an aptamer may be one or plural, but the plural is more sensitive as a sensor. Goes up.
  • the substance to be examined is a nucleic acid in the sixth embodiment, whereas the second double-stranded nucleic acid is not bound to a member in the seventh embodiment.
  • a target molecule is detected by examining a substance bound to the first double-stranded nucleic acid binding substance bound to the member or a distant substance. Therefore, it is possible to remove or recover a substance separated from the first double-stranded nucleic acid binding substance, detect a substance bound to the first double-stranded nucleic acid binding substance or a separated substance, What has been described in the embodiment can be said to be the same in the seventh embodiment.
  • a sensor device and method using the aptamer according to the present invention which can confirm the presence of a target molecule by detecting a second double-stranded nucleic acid binding substance, is described. did.
  • the sensor device according to the seventh embodiment is excellent in versatility and reusability because the devices used in the first to fifth embodiments can be used as they are. Furthermore, when the aptamer selected in the first to fifth embodiments is used, when the target molecule is bound to the aptamer, an experiment for confirming that the aptamer is separated from the double-stranded binding substance, etc.
  • the aptamer can be used for the sensor device as it is. Furthermore, if a conductive substance is used for the member, the first double-stranded nucleic acid binding substance, and the second double-stranded nucleic acid binding substance, the presence of the target molecule can be easily detected by current measurement.
  • the present invention can be widely used for selecting aptamers that are nucleic acids that specifically bind to molecules.

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention vise à développer un procédé et un appareil pour sélectionner un aptamère (un acide nucléique capable de se lier de façon spécifique à une molécule), qui peuvent être obtenus par des processus simples et qui ont une large utilité. Le procédé de sélection d'un aptamère est un procédé de sélection d'un aptamère (un acide nucléique capable de se lier de façon spécifique à une molécule) et comporte les étapes suivantes : une première étape de liaison d'une substance de liaison à un acide nucléique double brin qui peut se lier de façon spécifique à une partie double brin dans un acide nucléique à une partie double brin dans un premier acide nucléique simple brin (un candidat aptamère), la partie double brin étant formée par l'hybridation d'au moins une partie dans la séquence du premier acide nucléique simple brin ; une deuxième étape de liaison du premier acide nucléique simple brin à une molécule à laquelle le premier acide nucléique simple brin peut se lier de façon spécifique, et une troisième étape de dissociation du premier acide nucléique simple brin lié à la molécule à partir de la substance de liaison à un acide nucléique double brin.
PCT/JP2009/056792 2008-04-01 2009-04-01 Procédé et appareil de sélection d'un aptamère et procédé de détection utilisant l'aptamère Ceased WO2009123255A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029224A1 (fr) * 2010-09-01 2012-03-08 日本電気株式会社 Procédé pour détecter une substance cible, ensemble d'aptamères utilisé pour celui-ci, capteur, et dispositif
WO2012086599A1 (fr) * 2010-12-20 2012-06-28 Ｎｅｃソフト株式会社 Outil de détection et système de détection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MORSE,D.P.: "Direct selection of RNA beacon aptamers", BIOCHEM BIOPHYS RES COMMUN, vol. 359, no. 1, 2007, pages 94 - 101 *
RAJENDRAN, M. ET AL.: "In vitro selection of molecular beacons", NUCLEIC ACIDS RES, vol. 31, no. 19, 2003, pages 5700 - 13 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029224A1 (fr) * 2010-09-01 2012-03-08 日本電気株式会社 Procédé pour détecter une substance cible, ensemble d'aptamères utilisé pour celui-ci, capteur, et dispositif
CN103108952A (zh) * 2010-09-01 2013-05-15 日本电气株式会社 检测目标物质的方法以及用于所述方法中的适体组、传感器和装置
JP5803923B2 (ja) * 2010-09-01 2015-11-04 日本電気株式会社 標的物質の検出方法、それに用いるアプタマーセット並びにセンサ及び装置
WO2012086599A1 (fr) * 2010-12-20 2012-06-28 Ｎｅｃソフト株式会社 Outil de détection et système de détection
JPWO2012086599A1 (ja) * 2010-12-20 2014-05-22 Ｎｅｃソフト株式会社 検出用具および検出システム
US10295531B2 (en) 2010-12-20 2019-05-21 Nec Solutions Innovators, Ltd. Detection instrument, and detection system

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JPWO2009123255A1 (ja) 2011-07-28
JP5317221B2 (ja) 2013-10-16

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