[go: up one dir, main page]

WO2009157219A1 - Procédé de détection de manière électrochimique d'acide nucléique avec l'utilisation d'un conjugué d'adn - Google Patents

Procédé de détection de manière électrochimique d'acide nucléique avec l'utilisation d'un conjugué d'adn Download PDF

Info

Publication number
WO2009157219A1
WO2009157219A1 PCT/JP2009/054542 JP2009054542W WO2009157219A1 WO 2009157219 A1 WO2009157219 A1 WO 2009157219A1 JP 2009054542 W JP2009054542 W JP 2009054542W WO 2009157219 A1 WO2009157219 A1 WO 2009157219A1
Authority
WO
WIPO (PCT)
Prior art keywords
host compound
reporter molecule
modified oligonucleotide
base sequence
cyclodextrin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2009/054542
Other languages
English (en)
Japanese (ja)
Inventor
敏博 井原
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kumamoto University NUC
Original Assignee
Kumamoto University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kumamoto University NUC filed Critical Kumamoto University NUC
Priority to JP2010517792A priority Critical patent/JPWO2009157219A1/ja
Publication of WO2009157219A1 publication Critical patent/WO2009157219A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates to a method for electrochemical detection of nucleic acid using a DNA conjugate.
  • Non-patent Document 1 S. Takenaka, Y. Uto, H. Kondo, T. Ihara, M. Takagi, Anal. Biochem., 1994, vol. 218, pp. 436-443;
  • Non-patent literature 2 T. Ihara, Y. Maruo , S. Takenaka, M. Takagi, Nucleic Acids Res., 1996, vol. 24, pp. 4273-4280).
  • Non-patent Document 3 T. Ihara, M.
  • Non-Patent Document 5 C.ooE. Immoos, .S. J. Lee, M. W. Grinstaff, J. Am. Chem. Soc., 2004, vol. 126, pp. 10814-10815. This is because the structure of the conjugate immobilized on the electrode changes due to hybridization with the target DNA (hairpin ⁇ duplex), and the distance between Fc and the electrode changes accordingly. Based on a changing mechanism. However, this system still requires DNA immobilization.
  • Non-Patent Document 6 S. Takenaka, T. Ihara, M. Hamano, M. Takagi, J. Chem. Soc., Chem. Commun., 1990, vol. 1990, pp. 1271-1273
  • the present invention provides the following modified oligonucleotide, electrochemical gene detection kit, and electrochemical gene detection method.
  • the host compound is any one of ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, derivatives thereof, calixarenes, cyclophanes, and cucurbiturils. Or the modified oligonucleotide as described in (2).
  • An electrochemical gene detection kit comprising a first modified oligonucleotide having a host compound bound to the 5 ′ end, and a second modified oligonucleotide having a reporter molecule (guest compound) bound to the 3 ′ end.
  • the first modified oligonucleotide includes a base sequence complementary to the extent that it can specifically hybridize to the base sequence of a desired site of the target polynucleotide
  • the second modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of the site adjacent to the 3 ′ end of the desired site
  • the kit for electrochemical gene detection wherein the reporter molecule can form an inclusion complex with the host compound, and the electrochemical signal changes when the reporter molecule is included in the host compound.
  • An electrochemical gene detection kit comprising a first modified oligonucleotide having a host compound bound to the 3 ′ end, and a second modified oligonucleotide having a reporter molecule (guest compound) bound to the 5 ′ end.
  • the first modified oligonucleotide includes a base sequence complementary to the extent that it can specifically hybridize to the base sequence of a desired site of the target polynucleotide
  • the second modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of the site adjacent to the 5 ′ end of the desired site
  • the kit for electrochemical gene detection wherein the reporter molecule can form an inclusion complex with the host compound, and the electrochemical signal changes when the reporter molecule is included in the host compound.
  • the above (6) wherein the host compound is any one of ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, derivatives thereof, calixarenes, cyclophanes, and cucurbiturils. Or the kit as described in (7).
  • the first modified oligonucleotide having the host compound bound to the 5 ′ end and the second modified oligonucleotide having the reporter molecule (guest compound) bound to the 3 ′ end are mixed with the sample solution containing the target polynucleotide.
  • An electrochemical gene detection method comprising: The first modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of a desired site of the target polynucleotide, The second modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of the site adjacent to the 3 ′ end of the desired site, An electrochemical gene detection method, wherein the reporter molecule can form an inclusion complex with the host compound, and the electrochemical signal changes when the reporter molecule is included in the host compound.
  • the first modified oligonucleotide having the host compound bound to the 3 ′ end and the second modified oligonucleotide having the reporter molecule (guest compound) bound to the 5 ′ end are mixed with the sample solution containing the target polynucleotide.
  • An electrochemical gene detection method comprising: The first modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of a desired site of the target polynucleotide, The second modified oligonucleotide contains a base sequence complementary to the extent that it can specifically hybridize to the base sequence of the site adjacent to the 5 ′ end of the desired site, An electrochemical gene detection method, wherein the reporter molecule can form an inclusion complex with the host compound, and the electrochemical signal changes when the reporter molecule is included in the host compound.
  • a reporter molecule guest compound
  • An electrochemical gene detection kit comprising a modified oligonucleotide having a host compound bound to the 5 ′ end and a reporter molecule (guest compound) bound to the 3 ′ end,
  • the modified oligonucleotide includes a base sequence that is complementary to the extent that it can specifically hybridize to the base sequence of the desired site of the target polynucleotide,
  • the reporter molecule can form an inclusion complex with the host compound, and its electrochemical signal changes depending on whether or not it is included in the host compound. Detection kit.
  • An electrochemical gene detection kit comprising a modified oligonucleotide in which a host compound is bound to the 3 ′ end and a reporter molecule (guest compound) is bound to the 5 ′ end,
  • the modified oligonucleotide includes a base sequence that is complementary to the extent that it can specifically hybridize to the base sequence of the desired site of the target polynucleotide,
  • the reporter molecule can form an inclusion complex with the host compound, and its electrochemical signal changes depending on whether or not it is included in the host compound. Detection kit.
  • the host compound is any one of ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, derivatives thereof, calixarenes, cyclophanes, and cucurbiturils. Or the kit according to (29). (31) The kit according to (28) or (29), wherein the host compound is ⁇ -cyclodextrin or a derivative thereof. (32) The above-mentioned (28), wherein the reporter molecule is an electrochemically active small molecule selected from the group consisting of metallocene, metal complex, anthraquinone, viologen, methylene blue, flavin, fullerene, donomycin, and derivatives thereof.
  • the reporter molecule can form an inclusion complex with the host compound, and its electrochemical signal changes depending on whether or not it is included in the host compound. Detection method.
  • nucleotide sequence that is complementary to the nucleotide sequence of a desired site of the target polynucleotide and capable of specifically hybridizing, the host compound at the 3 ′ end and the reporter molecule (guest compound) at the 5 ′ end Mixing the bound modified oligonucleotide with a sample solution containing the target polynucleotide, and detecting a change in the electrochemical signal of the reporter molecule;
  • An electrochemical gene detection method comprising: The reporter molecule can form an inclusion complex with the host compound, and its electrochemical signal changes depending on whether or not it is included in the host compound. Detection method.
  • DNA conjugate of the present invention for example, ⁇ -cyclodextrin (which may be abbreviated as “ ⁇ CyD” in this specification) modified DNA conjugate as one embodiment
  • the properties can be controlled and used for gene detection.
  • ⁇ CyD can be arranged at a desired position on the template DNA (target DNA) by appropriately selecting the base sequence of the conjugate DNA.
  • a DNA conjugate is prepared by modifying a relatively hydrophobic reporter molecule (signaling molecule) having the property of being encapsulated by ⁇ CyD.
  • the reporter molecule and ⁇ CyD can be arranged to face each other on the template.
  • any molecule can be used as long as its signal is changed by inclusion in ⁇ CyD.
  • DNA conjugates in which both ends of one DNA are modified with ⁇ CyD and a reporter molecule can also be used for gene analysis.
  • Fc is included in ⁇ CyD in a tandem double-stranded complex, Utilizing the fact that the electron transfer speed is remarkably reduced by being shielded from the bulk (FIG. 4).
  • ⁇ CyD has a function corresponding to a quencher in the fluorescence method, and therefore the system used in the spectroscopic method can be transferred to one using the electrochemical method.
  • DNA modified with a fluorescent substance and a quenching substance at both ends is well known as a molecular beacon (MB), but as one embodiment of the present invention, both ends are modified with Fc and ⁇ CyD instead of the fluorescent substance and the quenching substance.
  • the DNA conjugate can be used as an electrochemical molecular beacon (ECMB) of a type in which the current value increases with a structural change from a hairpin type to a double strand (FIG. 6).
  • a highly sensitive electrochemical gene detection method a modified oligonucleotide used therefor, and a kit are provided.
  • the electrochemical gene detection method of the present invention it is possible to discriminate between individual differences (SNPs) at a single base level, which has been difficult with conventional methods using small molecules such as intercalators.
  • the method of the present invention is advantageous in that it does not require immobilization of oligonucleotides on a solid phase, which was necessary in the conventional method using an Fc-DNA conjugate.
  • the electrochemical method of the present invention is advantageous in that it is inexpensive and can be miniaturized according to the application.
  • the present invention is useful as a basic technique particularly in gene detection / analysis techniques in the biotechnology field.
  • the modified oligonucleotide ferrocene (Fc) of the present invention is well known as an electrochemically active molecule, but its size fits well into the pores of ⁇ -cyclodextrin ( ⁇ CyD) to form an inclusion complex To do.
  • ⁇ CyD ⁇ -cyclodextrin
  • two DNA conjugates modified with Fc and ⁇ CyD at the 3 ′ and 5 ′ ends of two DNAs complementary to adjacent sites of the target DNA, respectively, 3′Fc ⁇ Oligodeoxynucleotide (ODN) and 5′CyD-ODN are synthesized as probes.
  • ODN Oligodeoxynucleotide
  • 5′CyD-ODN 5′CyD-ODN
  • the present invention in one embodiment, provides a modified oligonucleotide conjugated with ⁇ -cyclodextrin at one end.
  • ⁇ -cyclodextrin is attached to the 5 'end of the oligonucleotide.
  • the base sequence of this oligonucleotide is configured to be complementary to the base sequence of the desired site of the target polynucleotide.
  • this complementarity does not need to be perfect (100%), and it is sufficient if it is complementary to the extent that it can specifically hybridize to the desired site. Therefore, the degree of complementarity is appropriately adjusted according to the purpose. You can do it.
  • oligonucleotide hybridization varies (or can be adjusted) depending on the temperature, salt concentration, and the like at the time of hybridization.
  • Hybridization at relatively low salt concentrations and / or high temperature conditions eg, salt concentrations from about 0.02 mol / l to about 0.15 mol / l and temperatures from about 50 ° C. to about 70 ° C.
  • salt concentrations from about 0.02 mol / l to about 0.15 mol / l and temperatures from about 50 ° C. to about 70 ° C.
  • the oligonucleotide may be an oligoribonucleotide whose sugar moiety is composed of ribose or an oligodeoxyribonucleotide whose sugar moiety is composed of deoxyribose, and is preferably an oligodeoxyribonucleotide.
  • the lower limit of the length of this oligonucleotide is about 2 nucleotides, preferably 3 nucleotides, more preferably 4 nucleotides, more preferably 5 nucleotides, still more preferably 6 nucleotides, and most preferably 7 nucleotides or more.
  • the upper limit is about 100 nucleotides, preferably about 80 nucleotides, more preferably about 60 nucleotides, more preferably about 40 nucleotides, still more preferably about 30 nucleotides, and most preferably about 20 nucleotides or more. small.
  • the length of the oligonucleotide is from about 5 nucleotides to about 20 nucleotides, but is not limited thereto.
  • the oligonucleotide used in the present invention can be synthesized with a desired length and base sequence using an automatic synthesizer or the like.
  • the above modified oligonucleotide (first modified oligonucleotide) can be used for electrochemical detection of a gene together with a second modified oligonucleotide having a reporter molecule bound to one end.
  • the second modified oligonucleotide has a base complementary enough to specifically hybridize to the base sequence of the site adjacent to the desired site on the target polynucleotide to which the first modified oligonucleotide hybridizes. Contains an array.
  • the reporter molecule is attached to the 3 ′ end of the second polypeptide and the second oligonucleotide is hybridized to the first oligonucleotide. And a base sequence complementary to the base sequence of the site adjacent to the 3 ′ end of the desired site on the target polynucleotide. If ⁇ CyD is attached to the 3 ′ end of the first oligonucleotide, the reporter molecule is attached to the 5 ′ end of the second polypeptide and the second oligonucleotide is hybridized to the first oligonucleotide.
  • ⁇ CyD and the reporter molecule can be arranged to face each other.
  • the above reporter molecule opposed to ⁇ CyD can form an inclusion complex with ⁇ -cyclodextrin.
  • the inclusion complex is formed, the electrochemical signal of the reporter molecule is shielded and attenuated. By detecting this change in signal intensity, hybridization between the target polynucleotide and the modified oligonucleotide of the present invention can be confirmed.
  • the “inclusion complex” is also referred to as a clathrate compound, which is a kind of molecular complex, and one chemical species creates a one- to three-dimensional molecular scale space.
  • This refers to a compound that is produced by the inclusion of the other chemical species with the first requirement that the size and shape conform to the space (Oki et al., “Chemical Encyclopedia”, 1st edition, Tokyo Chemical Doujin 1989).
  • a chemical species that provides space is called a host, and an enclosed species is called a guest.
  • a typical example of an inclusion complex used in the present invention comprises ⁇ -cyclodextrin as a host and ferrocene as a guest.
  • the combination of the inclusion complexes that can be used in the present invention is not limited to this, and any combination may be used as long as the electrochemical signal of the guest compound detected before and after the formation of the inclusion complex changes.
  • the reporter molecule (guest compound in the present invention) is not particularly limited as long as its electrochemical signal changes when it is included in ⁇ -cyclodextrin or other host compound.
  • reporter molecules used in the present invention include electrochemically active small molecules such as electrochemically active metallocenes such as ferrocene, metal complexes, anthraquinone, viologen, methylene blue, flavin, fullerene, donomycin, and derivatives thereof. Can be mentioned.
  • the host compound used in the present invention is not particularly limited as long as it can provide a space in which the reporter molecule can be included and can shield its electrochemical activity when the reporter molecule is incorporated.
  • Examples of the host compound used in the present invention include cyclodextrin (CyD) (for example, ⁇ -cyclodextrin ( ⁇ CyD), ⁇ -cyclodextrin ( ⁇ CyD), ⁇ -cyclodextrin ( ⁇ CyD) and the like) or derivatives thereof , Calixarenes, cyclophanes, and cucurbiturils.
  • the calixarenes, cyclophanes, and cucurbiturils include calixarene and derivatives thereof, cyclophanes and derivatives thereof, and cucurbiturils and derivatives thereof, respectively.
  • Examples of suitable combinations of the above electrochemically active guest compound and host compound include CyD-ferrocene (DR van Staveren, N. Metzler-Nolte, Chem. Rev., 104, 5931-5985 (2004). ), CyD-metallocene (F. Hapiot, S. Tilloy, E. Monflier, Chem. Rev., 106, 767-781 (2006)), CyD-anthraquinone (X.-J. Dang, J. Ton, H. -L. Li, J. Incl. Phenom., 24, 275-286 (1996), Cucurbituril-ferrocene (D. Sobrancingh, A. E.
  • CyD- flavin include many examples such CyD- fullerenes.
  • CyD in general “Cyclodextrins and Their Complexes”, H.C. Dodziuk Ed. , Wiley-VCH (2006).
  • the host compound-modified oligonucleotide of the present invention in which the host compound is bonded to one end of the oligonucleotide is a coupling between an activated host compound and an oligonucleotide whose primary amino group or thiol group is chemically modified, or It can be prepared by DNA synthesis using an amidite reagent based on a host compound derivative.
  • An oligonucleotide with a reporter molecule (guest compound) attached to one end is coupled to an activated reporter molecule with an oligonucleotide chemically modified with a primary amino group or thiol group at the end, or an amidite based on a reporter molecule It can be prepared by DNA synthesis using a reagent.
  • the target polynucleotide refers to a polynucleotide (eg, target gene) to be detected in the gene detection method of the present invention, and may be DNA or RNA, but is preferably DNA.
  • the present invention provides a modified oligonucleotide in which ⁇ -cyclodextrin and a reporter molecule are bound to both ends of one oligonucleotide.
  • ⁇ -cyclodextrin is attached to the 5 ′ end
  • the reporter molecule is attached to the 3 ′ end
  • the reporter gene is attached to the 5 ′ end.
  • the oligonucleotide is configured to include a base sequence that is complementary to the extent that it can specifically hybridize to the base sequence of the desired site of the target polynucleotide.
  • the reporter molecule can form an inclusion complex with ⁇ -cyclodextrin, and its electrochemical signal changes depending on whether it is included in ⁇ -cyclodextrin or not.
  • host compounds such as ⁇ -cyclodextrin, ⁇ -cyclodextrin, or derivatives thereof, calixarenes, cyclophanes, and cucurbiturils can be used.
  • electrochemically active small molecules such as metallocenes including ferrocene, metal complexes, anthraquinones, viologens, methylene blue, flavins, fullerenes, donomycin, and derivatives thereof can be used.
  • modified oligonucleotide configured in this manner is not hybridized to the target polynucleotide, ⁇ CyD at both ends and the reporter molecule form an inclusion complex, and take a hairpin-like form (stem loop structure) .
  • the loop structure is released to take an extended form.
  • this form of modified oligonucleotide hybridizes to the target polynucleotide, an increase in electrochemical signal from the reporter molecule is detected.
  • DNA modified with a fluorescent substance and a quenching substance at both ends is well known as a molecular beacon (MB).
  • a DNA in which Fc and ⁇ CyD are modified instead of the fluorescent substance and the quenching substance can be used as an electrochemical molecular beacon (ECMB) of a type in which the current value increases with a structural change from a hairpin type to a double strand (FIG. 6).
  • ECMB electrochemical molecular beacon
  • the present invention provides an electrochemical gene detection method using the modified oligonucleotide of the present invention.
  • the electrochemical gene detection method of the present invention comprises a step of mixing the modified oligonucleotide of the present invention with a sample solution containing a target polynucleotide, and then detecting a change in the electrochemical signal of the reporter molecule.
  • the reporter molecule (guest compound) is ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, derivatives thereof, calixarenes, cyclophanes
  • the electrochemical signal of the reporter molecule is attenuated by forming an inclusion complex with a host compound such as Cucurbiturils, or the reporter molecule is electrochemically shielded in the case of molecular beacon type modified oligonucleotides Is released and the signal increases.
  • the target polynucleotide is detected.
  • the presence of nucleotides can be detected.
  • the electrochemical signal emitted from the reporter molecule is obtained by, for example, placing a mixed solution of ⁇ CyD-modified oligonucleotide and reporter molecule-modified oligonucleotide into a three-electrode electrolytic cell comprising a working electrode, a counter electrode, and a reference electrode, Is added, followed by various voltammetry such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV). Alternatively, measurement can be performed with higher sensitivity by introducing the previous sample into a flow type electrochemical detector. Examples of the sample containing the target polynucleotide include, but are not limited to, a sample obtained by amplifying a cell extract by PCR. 3. Gene detection kit comprising the modified oligonucleotide of the present invention
  • the present invention provides an electrochemical gene detection kit comprising the modified oligonucleotide of the present invention.
  • the kit of the present invention can be used for carrying out the electrochemical gene detection method of the present invention.
  • the kit of the present invention may contain an instruction, a container, a label and the like describing the method of use as necessary.
  • Example 1 Preparation of 3'Fc-ODN and 5'CyD-ODN Ferrocenecarboxylic acid was reacted with N-hydroxysuccinimide to obtain an active ester of ferrocenecarboxylic acid.
  • a 3′Fc-ODN was obtained by coupling an oligonucleotide having a primary amino group introduced at the 3 ′ end with a large excess of an active ester of ferrocenecarboxylic acid in a buffer and DMSO mixed solvent. Purification by HPLC and identification by MALDI-TOF / MS.
  • One of the primary hydroxyl groups of ⁇ -cyclodextrin was tosylated and then converted to a thiol group.
  • the thiol group was further activated with aldolthiol.
  • An oligonucleotide having a thiol group introduced at the 5 'end was coupled with the previously activated ⁇ -cyclodextrin to obtain 5'CyD-ODN. Purification by HPLC and identification by MALDI-TOF / MS.
  • FIG. 1A and B show the structures of A) 3'Fc-ODN and B) 5'CyD-ODN, respectively.
  • FIG. 1A shows a 15-mer DNA modified with Fc at the 3 ′ end
  • FIG. 1B shows a 7-mer modified with ⁇ CyD at the 5 ′ end.
  • Both conjugates are complementary to adjacent sites in the target DNA. The base sequences of both conjugates are designed so that the modified molecules face each other when they form a tandem duplex with the target.
  • Example 2 Melting experiment of tandem double strand with target DNA Melting experiment was performed using the tandem double strand prepared in Example 1.
  • 5′CyD-ODN, 3′Fc-ODN, and target DNA were dissolved in a buffer solution (10 mM phosphate buffer (pH 7.0), 1M NaCl) so as to be 1 ⁇ M, and two tandems consisting of three members A chain (3′Fc-ODN / 5′CyD-ODN / target) was formed.
  • the absorbance at 260 nm was measured while slowly raising the temperature of the solution from 0 ° C. (0.2 ° C./min or less).
  • thermodynamic parameters K, ⁇ G, ⁇ H, ⁇ S
  • FIG. 2 is a diagram showing the melting behavior of a tandem duplex. Low temperature melting indicates octamer 5'CyD-ODN and high temperature melting indicates 15mer unmodified DNA or 3'Fc-ODN. When nothing was modified at the end of the adjacent 15-mer (dotted line), the melting temperature was 18.7 ° C., and when Fc was modified (solid line), it was 37.4 ° C.
  • Example 3 Measurement of electrochemical signal (procedure) Electrochemical measurement was performed in a phosphate buffer (10 mM phosphate buffer (pH 7), 0.5 M KCl). Cyclic voltammetry was performed using the working electrode and gold as the counter electrode and Ag / AgCl as the reference electrode. Only 3′Fc-ODN was measured for 3′Fc-ODN / 5′CyD-ODN, 3′Fc-ODN / target, and tandem double-stranded 3′Fc-ODN / 5′CyD-ODN / target.
  • FIG. 3 is a cyclic voltammogram of the DNA complex.
  • the current values of the 3′Fc-ODN / target and 3′Fc-ODN / 5′CyD-ODN systems were almost the same as when 3′Fc-ODN alone was used.
  • a tandem duplex (3′Fc-ODN / 5′CyD-ODN / target) was formed, and the current was remarkably suppressed only when Fc and ⁇ CyD were in contact with each other on the target.
  • FIG. 4 schematically shows how Fc is included in ⁇ CyD on a tandem double-stranded complex.
  • FIG. 4 shows one possible structure of a 3'Fc-ODN / target / 5'CyD-ODN tandem duplex. Since Fc is included in ⁇ CyD and shielded from the outside world, the electron transfer reaction with the electrode is remarkably suppressed.
  • ⁇ CyD can be expected to have a function corresponding to a quenching molecule in the fluorescence method.
  • Example 4 Application of the modified oligonucleotide of the present invention to gene analysis (part 1) Based on the results of Example 3, the TPMT gene (thiopurine methyltransferase: a gene encoding an enzyme involved in drug metabolism. The dose of the anticancer drug mercaptopurine is determined by the type of SNP (single nucleotide polymorphism) base). The gene sequence including the hot spot was chemically synthesized, and the possibility of application to gene analysis was examined.
  • TPMT gene thiopurine methyltransferase: a gene encoding an enzyme involved in drug metabolism.
  • the dose of the anticancer drug mercaptopurine is determined by the type of SNP (single nucleotide polymorphism) base).
  • SNP single nucleotide polymorphism
  • FIG. 5 shows the square wave bontammetry of the composite.
  • the template DNA target DNA
  • GC 3′Fc-ODN
  • the electron transfer is remarkably suppressed due to the inclusion of ⁇ CyD to form a stable double strand.
  • DNA with a single base substitution was used as a template, inclusion was not complete, and a large current value was shown.
  • FIGS. 6A and 6B are diagrams showing the operation principle of (A) a conventional molecular beacon (MB) and (B) the electrochemical molecular beacon (ECMB) of the present invention.
  • an oligonucleotide in which Fc and ⁇ CyD are modified at both ends usually has a hairpin structure with a complementary quantifier at the end forming a stem.
  • Fc is included in ⁇ CyD and its electrochemical activity Is suppressed.
  • the target is added, the hairpin structure is eliminated and a double strand with the target is formed.
  • Fc is exposed and regains its original electrochemical activity. That is, in the electrochemical method, ⁇ CyD can be used as a unit that plays the same role as the quencher in the conventional fluorescence method.
  • Electrochemical methods are more sensitive than spectroscopic methods, equipment used is inexpensive, and can be miniaturized according to the application.
  • the present invention provides a new gene detection method based on such an electrochemical method, and is useful as a basic technology in the field of biotechnology, particularly in gene detection / analysis technology.
  • the structures of DNA conjugates (A) 3'Fc-ODN and (B) 5'CyD-ODN are shown.
  • the melting behavior of a tandem duplex is shown. It is a cyclic voltammogram of a DNA complex.
  • One of the possible structures of the tandem duplex of 3'Fc-ODN / target / 5'CyD-ODN is shown.
  • the square wave bontammetry of a composite is shown. It is a figure which shows the principle of operation of (A) conventional molecular beacon (MB) and (B) the electrochemical molecular beacon (ECMB) of this invention.
  • MB conventional molecular beacon
  • ECMB electrochemical molecular beacon

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Materials Engineering (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

La présente invention concerne une technique de détection aisée et précise d'un gène sans avoir recours à l'immobilisation de l'ADN et ainsi de suite. L'invention concerne également un oligonucléotide modifié ayant un composé hôte tel qu'une cyclodextrine lié à une extrémité de celui-ci et contenant une séquence de bases qui est suffisamment complémentaire pour qu'il puisse s'hybrider spécifiquement à la séquence de bases à un site souhaité d'un polynucléotide cible.
PCT/JP2009/054542 2008-06-27 2009-03-10 Procédé de détection de manière électrochimique d'acide nucléique avec l'utilisation d'un conjugué d'adn Ceased WO2009157219A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2010517792A JPWO2009157219A1 (ja) 2008-06-27 2009-03-10 Ｄｎａコンジュゲートを利用した核酸の電気化学的検出法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008168127 2008-06-27
JP2008-168127 2008-06-27

Publications (1)

Publication Number Publication Date
WO2009157219A1 true WO2009157219A1 (fr) 2009-12-30

Family

ID=41444291

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2009/054542 Ceased WO2009157219A1 (fr) 2008-06-27 2009-03-10 Procédé de détection de manière électrochimique d'acide nucléique avec l'utilisation d'un conjugué d'adn

Country Status (2)

Country Link
JP (1) JPWO2009157219A1 (fr)
WO (1) WO2009157219A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8454820B2 (en) * 2009-09-09 2013-06-04 National Institute Of Advanced Industrial Science And Technology Electrochemical molecular recognition probes
CN110330665A (zh) * 2019-07-24 2019-10-15 南京融众环境工程研究院有限公司 一种七元瓜环与磺化硫杂杯[4]芳烃基超分子聚合物及制备方法和应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"87th Annual Meeting on Chemical Society of Japan in Spring -Koen Yokoshu II", 12 March 2007, article YU MUTO ET AL.: "Pyrene to Cyclodextrin de Shushoku shita Oligonucleotide o Mochiiru Suiyosei Bunshi Sensor no Kaihatsu", pages: 1350 *
"88th Annual Meeting on Chemical Society of Japan in Spring -Koen Yokoshu II", 12 March 2008, article TOSHIHIRO IHARA ET AL.: "§-Cyclodextrin Shushoku DNA o Mochiiru Denki Kagakuteki Idenshi Kaisekiho ni Kansuru Kiso Kenkyu", pages: 811 *
"88th Annual Meeting on Chemical Society of Japan in Spring -Koen Yokoshu II", article TOSHIHIRO IHARA ET AL.: "§-Cyclodextrin Shushoku DNA to Enki Shikibetsuno o Yusuru Keikosei Ligand o Mochiiru Idenshi Kaiseki", pages: 811 *
"Dai 13 Kai Symposium on Polymers and Biosciences Koen Yoshishu", 31 July 2003, article YUICHI OYA ET AL.: "Cyclodextrin Ketsugo Oligo DNA Bunshi Soshikitai ni yoru Bunshi Ninshiki", pages: 75 - 76 *
"Dai 22 Kai Abstracts, Symposium on Biofunctional Chemistry", 10 September 2007, article AKINORI KUZUYA ET AL.: "Cyclodextrin Shushoku DNA no Gosei to Bio Nanotechnology eno Oyo", pages: 526 - 527 *
"Dai 22 Kai Abstracts, Symposium on Biofunctional Chemistry", 10 September 2007, article TOSHIHIRO IHARA ET AL.: "Enki Shikibetsuno o Motsu Keikosei Ligand to DNA Conjugate ni yoru Kyodoteki Kakusan Probing", pages: 216 - 217 *
BROWNE,K.A.: "Sequence-specific, self-reporting hairpin inversion probes", J AM CHEM SOC, vol. 127, no. 6, 2005, pages 1989 - 94 *
IHARA,T. ET AL.: "Cooperative DNA probing using a beta-cyclodextrin-DNA conjugate and a nucleobase-specific fluorescent ligand", J AM CHEM SOC, vol. 131, no. 4, 4 February 2009 (2009-02-04), pages 1386 - 7 *
IHARA,T. ET AL.: "Photochemical ligation of DNA conjugates through anthracene cyclodimer formation and its fidelity to the template sequences", J AM CHEM SOC, vol. 126, no. 29, 2004, pages 8880 - 1 *
KITAMURA,Y. ET AL.: "Asymmetric cooperativity in tandem hybridization of enantiomeric metal complex-tethered short fluorescent DNA probes", CHEM.COMMUN., 2005, pages 4523 - 4525 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8454820B2 (en) * 2009-09-09 2013-06-04 National Institute Of Advanced Industrial Science And Technology Electrochemical molecular recognition probes
CN110330665A (zh) * 2019-07-24 2019-10-15 南京融众环境工程研究院有限公司 一种七元瓜环与磺化硫杂杯[4]芳烃基超分子聚合物及制备方法和应用

Also Published As

Publication number Publication date
JPWO2009157219A1 (ja) 2011-12-08

Similar Documents

Publication Publication Date Title
Michel et al. Probing of nucleic acid structures, dynamics, and interactions with environment-sensitive fluorescent labels
Xiao et al. An electrochemical sensor for single nucleotide polymorphism detection in serum based on a triple-stem DNA probe
US6461820B1 (en) Electrochemical sensor using intercalative, redox-active moieties
Ho et al. Optical detection of DNA and proteins with cationic polythiophenes
Xu et al. Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations
US6071699A (en) Nucleic acid mediated electron transfer
US20030235854A1 (en) Methods for analyzing a nucleic acid
US20030077609A1 (en) Modified oligonucleotides and uses thereof
US7202037B2 (en) Electrochemical sensor using intercalative, redox-active moieties
CN112840035B (zh) 对多核苷酸进行测序的方法
AU2019445584B2 (en) Single-channel sequencing method based on self-luminescence
CA2257534A1 (fr) Transfert d'electrons induit par des acides nucleiques
Zavoiura et al. Quantum dot-PNA conjugates for target-catalyzed RNA detection
US20060228703A1 (en) Displacement assay for the detection of nucleic acid oligomer hybridization events
WO2009157219A1 (fr) Procédé de détection de manière électrochimique d'acide nucléique avec l'utilisation d'un conjugué d'adn
Sato et al. Electrochemical DNA detection using supramolecular interactions
EP3633049A1 (fr) Nouvelle sonde d'extinction de fluorescence pour mesurer un acide nucléique
CN110241175A (zh) 一种Tet2序列偏好性的研究方法
Schwechheimer et al. Synthesis of Dye‐Modified Oligonucleotides via Copper (I)‐Catalyzed Alkyne Azide Cycloaddition Using On‐and Off‐Bead Approaches
Chatelain et al. Synthesis of electrochemical probes for nucleic acid detection
Suprun et al. Artificial modified nucleotides for the electrochemical detection of nucleic acid amplification products
Leck Fluorescent nucleic acid probes for DNA sensing and imaging
JP3618768B2 (ja) 標的核酸の検出方法、プローブ、２本鎖核酸ハイブリッド内のミスマッチの検出方法及びプローブの製造方法
JPH11286616A (ja) 核酸染色剤、それを用いた二本鎖核酸の検出方法及び標的核酸の検出試薬
Ali The Control and Detection of Biomolecules Using Functional DNA

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09769929

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2010517792

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09769929

Country of ref document: EP

Kind code of ref document: A1