WO2005103247A1 - Sonde oligonucléotidique - Google Patents

Sonde oligonucléotidique Download PDF

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Publication number
WO2005103247A1
WO2005103247A1 PCT/JP2005/007666 JP2005007666W WO2005103247A1 WO 2005103247 A1 WO2005103247 A1 WO 2005103247A1 JP 2005007666 W JP2005007666 W JP 2005007666W WO 2005103247 A1 WO2005103247 A1 WO 2005103247A1
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Prior art keywords
group
compound
oligonucleotide probe
oligonucleotide
solution
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English (en)
Japanese (ja)
Inventor
Yasuo Komatsu
Naoshi Kojima
Ken Nonaka
Yumi Fujinawa
Junya Hashida
Isamu Muto
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Hitachi Software Engineering Co Ltd
National Institute of Advanced Industrial Science and Technology AIST
DNA Chip Research Inc
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Hitachi Software Engineering Co Ltd
National Institute of Advanced Industrial Science and Technology AIST
DNA Chip Research Inc
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Publication of WO2005103247A1 publication Critical patent/WO2005103247A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds

Definitions

  • the present invention relates to an oligonucleotide probe composed of a linker having an aromatic group and an oligonucleotide, a carrier having the oligonucleotide probe immobilized thereon, and a compound for synthesizing the oligonucleotide probe.
  • a functional group such as an amino group is introduced into an oligonucleotide via a linker (Coulleta 1-, Tetrahedron, vol. 27, 3991-3994 (19986) ) And Connolly, B. A., Nucleic Acids Res., Vol. 1, 5, 3 1 3 1—3 13 9 (1 98 7))).
  • linker the most frequently used linker with an amino group is a linker having 6 carbon atoms.
  • Oligonucleotide probes to which an amino group is bound via this linker are also commercially available as a probe library for a DNA chip (for example, MWG, Sigma dienosis, etc.).
  • a functional group such as an amino group or a mercapto group is introduced during the synthesis of the oligonucleotide.
  • reagents for introducing an amino group into an oligonucleotide include an amidite compound in which the amino group is protected with a trifluoroacetyl group or a monomethoxytrityl group.
  • the reagents as described above have a problem that the efficiency of introducing an amino group into an oligonucleotide is low.
  • the purified amino group-introduced oligonucleotide obtained above is immobilized on a carrier, sufficient reactivity of the amino group with the coating agent on the carrier surface is low, and sufficient oligonucleotide is immobilized on the carrier. Contains a high concentration of oligonucleotides I needed to spot the tide. As a result, a large number of oligonucleotides were required, which increased the price of the chip. Therefore, it has been desired to effectively immobilize the oligonucleotide probe on a carrier.
  • An object of the present invention is to provide an oligonucleotide probe which is firmly immobilized on a carrier, can be easily purified after synthesis, and efficiently retains a complementary target nucleic acid.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, synthesized a novel compound in which a reactive functional group required for immobilization to a carrier was linked to an aromatic group, and converted this into an oligonucleotide.
  • the inventors have found that the above-mentioned problems can be solved by introducing the present invention, and have completed the present invention.
  • the present invention includes the following inventions.
  • Oligonucleotide probe (Wherein A represents an oligonucleotide, D represents a divalent organic group having at least one aromatic group, and B represents a reactive functional group or a protected form thereof) Oligonucleotide probe.
  • R 2 is a general formula 3:
  • R 3 has the general formula 4:
  • R 4 is a direct bond or one (CH 2 ) (OCH 2 CH 2 ) q — O—,
  • R c and R 6 are each independently a direct bond or a group shown below:
  • n and t each independently represent an integer from 0 to 20,
  • n, w, i, and q each independently represent an integer of 1 to 20,
  • R 7 represents a hydrogen atom or a phosphate protecting group
  • L represents a divalent aromatic group, represents a hydrogen atom or a substituent
  • R 12 , 3 and R 14 each independently represent a direct bond or a substituted or unsubstituted divalent hydrocarbon group which may contain a hetero atom, and A ′ represents a hydroxyl group or an oligonucleotide. ).
  • OR 8 (where D 'represents a divalent organic group having at least one aromatic group, and B is Represents a reactive functional group or a protected form thereof, o represents an oxygen atom, P represents a phosphorus atom, R 8 represents a phosphate protecting group, R 9 and. Is an organic group, and may form a ring together with the nitrogen atom to which they are bonded.)
  • D ′ is a linear or branched, substituted or unsubstituted divalent hydrocarbon group optionally containing a heteroatom, and has at least one aromatic group. 15. The compound according to any one of 5).
  • L represents an aromatic group, represents a hydrogen atom or a substituent
  • R 2 , R 2 ′ and R 3 each independently represent a direct bond or a substituted or unsubstituted hetero atom.
  • R 2 is a general formula 3:
  • R 3 has the general formula 4: One HN NH (CH 2 ) t — R 6- (CH 2 ) w- (4)
  • R 4 is a direct bond or one (CH 2 ) i— ( ⁇ CH 2 CH 2 ) q — 0_, and R 5 and R 6 are each independently a direct bond or a group shown below:
  • n and t each independently represent an integer from 0 to 20,
  • n, w, i, and q each independently represent an integer of 1 to 20,
  • R 7 represents a hydrogen atom or a phosphate protecting group, HN
  • L is a divalent aromatic group, represents a hydrogen atom or a substituent
  • R 12, 1 13 ⁇ Pi 1 ⁇ 14 are each independently, comprise a direct bond or a hetero atom
  • R 15 represents a hydroxyl-protecting group
  • the oligonucleotide probe of the present invention has a high reaction efficiency to a carrier, the amount of oligonucleotide required for immobilization can be reduced.
  • the clear oligonucleotide probe has a high binding efficiency to the target nucleic acid, so that it can be detected with high sensitivity even if it has the same chain length as before.
  • the oligonucleotide probe of the present invention can be easily purified after synthesis, and can be purified by automation even when a plurality of types of probes are prepared.
  • FIG. 1 shows the structures of a conventional oligonucleotide probe and the oligonucleotide probe of the present invention.
  • FIG. 2 shows a method for synthesizing the amidite compound (Y2).
  • FIG. 3 shows a method for synthesizing the amidite compound (Y 3).
  • FIG. 4 shows a method for synthesizing the amidite compound (Y5, 6).
  • FIG. 5 shows a method for synthesizing the amidite compound ( ⁇ 7).
  • FIG. 6 shows a method for synthesizing the amidite compound ( ⁇ 8).
  • FIG. 7 shows a method for synthesizing the amidite compound ( ⁇ 9).
  • FIG. 8 shows the structure of the oligonucleotide probe (Xn—Sp).
  • Figure 9 shows the amount of oligonucleotide probe (Xn-Sp) immobilized on the glass substrate (a) and the reactivity with FITC (b: 50 nucleotide oligonucleotide, c: 25 nucleotide oligonucleotide). .
  • FIG. 10 shows the results of the hybridization in Example 6.
  • FIG. 11 shows a specific example of the intermediate compound.
  • FIG. 13 shows a method for synthesizing the amidite compound (Y10, Y11).
  • Figure 14 shows the X10—Sp25 sequence and the scheme of the X10-Sp25 deprotection reaction (a), and the results of reverse phase HP LC analysis of X10—Sp25 (b), And X10-Sp25 were subjected to acid treatment, and the results of reverse phase HP LC analysis (c) are shown.
  • the present invention will be described in detail.
  • the oligonucleotide probe of the present invention has a structure in which a linker having a reactive functional group and an aromatic group is bonded to an oligonucleotide. That is,
  • A represents an oligonucleotide
  • D represents a divalent organic group having at least one aromatic group
  • B represents a reactive functional group or a protected form thereof.
  • the oligonucleotide may be natural or synthetic, and includes a polynucleotide.
  • Oligonucleotides include nucleic acids such as DNA and RNA, double-stranded oligonucleotides, and oligonucleotide derivatives. PCR products are also included.
  • Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides have been converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides have been converted to N3'-P5, phosphoramidite bonds.
  • Oligonucleotide derivative oligonucleotide derivative in which report and phosphodiester bond in oligonucleotide are converted to peptide nucleoacid bond
  • oligonucleotide in which peracyl in oligonucleotide is substituted with C-15 propynyl peracyl Derivatives Oligonucleotide derivatives in which peracyl in oligonucleotides is substituted with C-5 thiazoleperacyl
  • Oligonucleotides Oligonucleotide derivative in which cytosine in otide is replaced by phenoxazine-modified cytosine, oligonucleotide derivative in which report in oligonucleotide is replaced by 2,1-propylribose, ribose in oligon
  • the number of bases of the oligonucleotide is usually 1 to 500, preferably 5 to 200, and more preferably 10 to 100.
  • B represents a reactive functional group or a protected form thereof.
  • Reactive functional groups are present on the carrier to which the oligonucleotide probe is to be immobilized.
  • a group capable of forming a covalent bond with an existing functional group such as an active ester group, an epoxy group, an aldehyde group, a carbodiimide group, an isothiocyanate group or a group capable of forming a covalent bond with an isocyanate group (for example, , An amino group, etc.), or a group capable of reacting with a maleimide group or a disulfide group (eg, a mercapto group, etc.).
  • a mercapto group and a diamino group are preferred.
  • the amino group is preferably a primary amino group.
  • the B may be a protected form of the reactive functional group.
  • the protected form means a form in which a hydrogen atom of the functional group is substituted with a protecting group.
  • the protecting group for the amino group is not particularly limited, but may be an acyl group, a carbamate group, a trialkylsilyl group, a phthalyl group, a carboxyalkylcarbonyl group, a tosyl group, a trifluoroacetyl group, a trityl group, and Disubstituted trityl groups.
  • Examples of the mono-substituted trityl group include, for example, a monoalkoxytrityl group, preferably a monoalkoxytrityl group having an alkoxy group having 1 to 4 carbon atoms, and more preferably a monomethoxytrityl group, Examples include an ethoxytrityl group, a monopropoxytrityl group, a monoisopropoxytrityl group and a monobutoxytrityl group.
  • a trityl group or a mono- or di-substituted trityl group has an advantage in that the synthesized oligonucleotide probe can be easily purified by a reversed-phase column because of its strong hydrophobicity.
  • an amino group when an amino group is protected with a trityl group or a mono- or di-substituted trityl group, it is necessary to react under a strong acidic condition for a long time in order to deprotect the amino group. Such conditions are not preferable in that they may damage the nucleic acid and require time.
  • the nucleic acid when the amino group is protected with a trityl group or a mono- or di-substituted trityl group, the nucleic acid may be damaged because it can be deprotected in a short time under mildly acidic conditions. No, and the deprotection time can be reduced. For example, deprotection can be performed by treating for 5 to 20 minutes in the presence of pH 2 to 6 or 5 to 80% by volume of acetic acid. '
  • the protective group for the mercapto group is not particularly limited, and examples thereof include a t-butyl group, an aralkyl group, a diphenylmethyl group, a triphenylmethyl group, and a dimethyl group.
  • the oligonucleotide is It is linked to a reactive functional group via a linker part represented by in the general formula 1.
  • D represents a divalent organic group having at least one aromatic group.
  • the divalent organic group is not particularly limited as long as it does not inhibit the binding property of the oligonucleotide probe to the carrier and the complementary binding to the target oligonucleotide, but preferably includes a hetero atom. It is a well-substituted or unsubstituted divalent hydrocarbon group.
  • a chain member of 250 preferably a chain member of 3 to 30, more preferably a chain member of 1 to 5, a chain member of 2 to 50, preferably a chain member of 3 to 3 0, more preferably an alkylene group having a chain member of 1 to 5, a chain member having a chain member of 2 to 50, preferably a chain member having a chain member of 3 to 30, more preferably a chain member having a chain member of 1 to 5
  • a divalent alicyclic hydrocarbon group having a chain member of 3 to 30, more preferably 1 to 10 is exemplified.
  • a part of carbon atoms may be substituted with a complex atom.
  • the hetero atom include an oxygen atom, a nitrogen atom, a sulfur atom, a silicon atom, and a phosphorus atom.
  • substituents examples include a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a substituted or unsubstituted amino group, a nitro group, a cyano group, and a substituted or unsubstituted carbon number of 1 to 1.
  • a halogen atom selected from fluorine, chlorine, bromine and iodine
  • a hydroxyl group a substituted or unsubstituted amino group, a nitro group, a cyano group
  • alkyl group substituted or unsubstituted alkenyl group having 1 to 10 carbon atoms
  • substituted or unsubstituted cycloalkyl group having 1 to 10 carbon atoms
  • substituted or unsubstituted alkoxy group having 1 to 10 carbon atoms
  • the linker portion D is preferably a group represented by the following general formula 2.
  • L represents an aromatic group
  • R 2 , R 2 ′ and R 3 each independently represent a direct bond or a substituent which may contain a hetero atom or Represents an unsubstituted divalent hydrocarbon group.
  • the substituent is the same as described above.
  • the Examples of the divalent hydrocarbon group include the same as those described above.
  • R 2 binds to the reactive functional group B
  • R 2 binds to the oxygen of the hydroxyl group or phosphate group at the 5 ′ end or 3 ′ end of the oligonucleotide.
  • R 2 ′ is a direct bond
  • the carbon atom in the general formula 2 is directly bonded to the hydroxyl or phosphate oxygen at the 5 ′ end or 3 ′ end of the oligonucleotide.
  • R 2 is preferably of the general formula 3:
  • R 3 is preferably of the general formula 4:
  • R 2 ' is preferably a direct bond or _R 2', Ru Oh one (CH 2) "one.
  • R 4 is bonded to the reactive functional group B, in R 3 , 1 ′ (CH 2 ) w — is bonded to the aromatic group L, and in R 2 , one (CH 2 ) is an oligo. Binds to nucleotides.
  • R 4 is preferably a direct bond or 1 (CH 2 ); — (OCH 2 CH 2 ) g — ⁇ -1.
  • R 5 , R 6 and R 2 ′′ are each independently a direct bond or a group shown below:
  • n and t each independently represent an integer of 0 to 20, preferably 0 to 10, more preferably 0 to 6, and still more preferably an integer of 0 to 3, n, w, i, q And j each independently represent an integer of 1 to 20, preferably 1 to 10, more preferably 1 to 6, and even more preferably 1 to 3.
  • 111 + 11 is usually 1 to 40, preferably 1 to 20, more preferably 1 to 5
  • t + w is usually 1 to 40, preferably 1 to 20, more preferably 1 to 5.
  • 5, i + q is usually 2 to 40, preferably 2 to 20, more preferably 2
  • R 7 represents a hydrogen atom or a phosphate protecting group.
  • the phosphate protecting group is not particularly limited, but preferred examples include a methyl group, a 2-cyanoethyl group, a 2-trimethylsilylethyl group, and a 4-oxypentyl group.
  • R 4 is preferably a direct bond.
  • R 5 is preferably
  • R 6 is preferably 1 O—.
  • examples of the aromatic group include a substituted or unsubstituted aromatic hydrocarbon group, a substituted or unsubstituted aromatic heterocyclic group, and a substituted or unsubstituted polycyclic aromatic group.
  • an aromatic group having high hydrophobicity is preferred.
  • the aromatic group does not include a nucleobase.
  • substituent of the aromatic group examples include a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a substituted or unsubstituted amino group, a nitro group, a cyano group, and a substituted or unsubstituted carbon atom.
  • substituted or unsubstituted aromatic hydrocarbon group examples include a substituted or unsubstituted monocyclic aromatic hydrocarbon group, specifically, phenyl, 2-fluorophenyl, 3-fluorophenyl, 4 -Phenol group, 2-phenyl phenyl group, 3-chloro phenyl group, 2,3-difluorophenyl group, 2,5-diphenylolenophenylene group, 3,5-difluorophenyl group, 2,3—dichloropheninole, 2,5—dichloropheninole, 3,5—dichloropheninole, 2-methinolephenine, 3-methylphenyl, 4-methylphenyl, 2-— Examples thereof include an ethylphenyl group, a 3-ethylphenyl group, and a 4-ethylphenyl group.
  • aromatic heterocyclic group examples include a pyridyl group, a pyridazinyl group, a pyrimidinyl group, a virazinyl group, a furyl group, a phenyl group, a pyrrolyl group, an imidazolyl group, a thiazolyl group, and an oxazolyl group.
  • substituted or unsubstituted aromatic heterocyclic group examples include 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl Group, 5-pyrimidinyl group, pyrazinyl group 2-furyl group, 3_furyl group, 2-thenyl group, 3-thenyl group, 2-pyrrolyl group, 3-pyrrolyl group, imidazolyl group, thiazolyl group, oxazolyl group, 2-Methyl-3-pyridyl group, 6-methyl-1-pyridyl group, 2-chloro-3-pyridyl group, 6-chloro-3-pyridyl group, 2-methoxy-13-pyridyl group, 6-methoxy-3 —Pyridyl group, 2,6-dichloro-3-pyridyl group, 2,6-dimethoxy-3
  • Polycyclic aromatic groups include naphthyl, phenanthryl, fluorenyl, anthryl, pyrenyl, indanyl, tetrahydronaphthyl, quinolyl, isoquinolyl, nnolinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, Examples include a phthalazinyl group, an indolyl group, an isoindolyl group, a benzofurinole group, a benzochenolinole group, an indazolinole group, a benzimidazoly / le group, and a benzothiazolyl group.
  • substituted or unsubstituted polycyclic aromatic group include: 1-naphthyl group, 2-naphthyl group, 4-indanyl group, 5-indanyl group, 5-tetrahydronaphthyl group, 6-tetrahydronaphthyl group, Quinolyl group, isoquinolyl group, cinnolinyl group, quinazolinyl group, quinoxalinyl group, naphthyridinyl group, phthaladyl group, indolyl group, isoindolyl group, benzofuryl group, benzochelyl group, indazolyl group, benzimidazolyl group, benzothiazolyl group, 2, 3-methylenedioxyphenyl group, 3,4-methylenedioxyphenyl group, 2,3-ethylenedioxyphenyl group, 3,4-ethylenedioxyphenyl group, 4 1-naphthyl group, 4-naphthy
  • Preferred substituted or unsubstituted polycyclic aromatic groups include a naphthyl group, an anthryl group, a pyrenyl group, a fluorenyl group, and a phenanthryl group.
  • an aromatic group is present in the main chain portion of D ', the aromatic group becomes a divalent form.
  • the aromatic group is preferably a monocyclic to pentacyclic aromatic hydrocarbon group.
  • a bicyclic to tetracyclic aromatic hydrocarbon group more specifically, a substituted or unsubstituted naphthyl group, anthryl group, phenanthryl group, fluorenyl group and pyrenyl group, particularly 1-naphthyl group 9 is an anthryl group.
  • the oligonucleotide probe of the present invention includes those having an additional oligonucleotide on the side chain of the linker portion D.
  • the further oligonucleotide may be the same or different from the other oligonucleotide.
  • examples of the oligonucleotide probe include those in which D in the general formula 1 is represented by the following general formula 2 ′.
  • L is a divalent aromatic group, represents a hydrogen atom or a substituent
  • Ru are each independently R 1 2, 1 13 ⁇ Pi 1 14, include a direct bond or a hetero atom
  • a ′ represents a hydroxyl group or an oligonucleotide.
  • R ii binds to the reactive functional group B
  • R 14 binds to the oxygen of the hydroxyl group or phosphate group at the 5 ′ end or 3 ′ end of the oligonucleotide.
  • R E 4 is a direct bond
  • general formula 2 'carbon atoms in the, 5 Origonukureo tides' end or 3' end directly binds to the oxygen of a hydroxyl group or phosphate group.
  • a ′ is an oligonucleotide
  • R i 3 binds to the hydroxyl or phosphate oxygen at the 5 ′ end or 3 ′ end of the oligonucleotide and R 13 is a direct bond
  • the general formula The carbon atom at 2 ' is directly bonded to the hydroxyl or phosphate oxygen at the 5' or 3 'end of the oligonucleotide.
  • Rue preferably has the general formula 3 ′:
  • R 12 is preferably of the general formula 4 ′
  • R 13 is preferably a direct bond or 1 (CH 2 ) ⁇ ⁇ _
  • R 14 is preferably a direct bond or 1 (CH 2 ) f —.
  • one (CH 2 ) a — is bonded to the reactive functional group B, and — (CH 2 ) e — is bonded to the aromatic group L in R 12 .
  • R 5 and R 6 are each independently a direct bond or a group shown below:
  • e and f each independently represent an integer of 20, preferably 1 to 10, More preferably represents an integer of 1 to 5, more preferably represents an integer of 1 to 3, each of a to d independently represents an integer of 0 to 20, preferably 0 to 10, more preferably ⁇ to 5 And more preferably an integer of 0 to 3.
  • a + b is usually 1-40, preferably 1-20, more preferably 1-5
  • c + d is usually 1-40, preferably 1-20, more preferably 1 to 5.
  • R 7 represents a hydrogen atom or a phosphate protecting group. Examples of the phosphate protecting group include, but are not particularly limited to, a methyl group, a 2-cyanoethyl group, a 2-trimethylsilylethyl group, and a 4-oxypentyl group.
  • Pi 1 1 4 is preferably a direct bond.
  • R 5 ′ is preferably one NH—CO— and R 6 ′ is preferably one CO—NH—.
  • the divalent aromatic group L is not particularly limited, but is preferably a highly hydrophobic divalent aromatic group, preferably a substituted or unsubstituted phenanthrylene group, fluorenylene group, naphthylene group, anthrylene group or pyrenylene group, Particularly, a naphthylene group and an anthrylene group are exemplified.
  • the invention also relates to intermediate compounds for synthesizing oligonucleotide probes.
  • the intermediate of the present invention has a structure in which the oligonucleotide portion is a phosphoramidite in the oligonucleotide probe. Is a compound having Therefore, in one embodiment, the intermediate compound of the present invention is represented by the following general formula 5.
  • B represents a reactive functional group or a protected form thereof
  • D ′ represents a divalent organic group having at least one aromatic group
  • O represents an oxygen atom
  • P represents a phosphorus atom
  • R 8 represents a phosphate protecting group
  • R 9 and 11 Is an organic group, and may form a ring together with the nitrogen atom to which they are bonded.
  • a methyl group, an ethyl group, a propyl group, a butyl group, an isoptyl group, a pentyl group, and an isopentyl group are exemplified.
  • R 9 and R 10 may be taken together with the nitrogen atom to which they are attached to form a ring group.
  • the ring is R 9 and i. May further contain a hetero atom in addition to the nitrogen atom to which is bonded.
  • Such a ring is preferably a ring having a ring member of 5 to 8, preferably 6, and includes, for example, a morpholine ring, a piperidine ring, a piperazine ring, a thiomorpholine ring and the like, and preferably a morpholine ring. .
  • the phosphate protecting group may be any one used in the phosphoramidite method, and is preferably a methyl group, a 2-cyanoethyl group, a 2-trimethylsilylethyl group, a 4-oxypentyl group, or the like. It may be included as a group.
  • the oligonucleotide probe of the present invention can be prepared using an intermediate compound in which D ′ in Formula 5 is equal to the above D.
  • an oligonucleotide probe of the general formula 1 in which D is represented by the general formula 2 can be prepared using an intermediate compound in which D ′ in the general formula 5 is equal to D.
  • the oligonucleotide probe of the general formula 1 in which D is represented by the general formula 2 ′ uses an intermediate compound in which the D ′ in the general formula 5 is represented by the following general formula 6.
  • L is a divalent aromatic group
  • 1 ⁇ represents a hydrogen atom or a substituent
  • 5 hydroxyl represents a protecting group
  • a protecting group used for protecting the ribose at the 5-position in DNA synthesis can be used, for example, an acetyl group, 5′—O—4, 4 ′ , 4,,, — tris (4-benzoyloxy) trityl group and dimethoxytrityl group, and a dimethoxytrityl group is preferred.
  • 1 is bonded to 8
  • R 14 is bonded to an oxygen atom. Specific examples of preferred intermediate compounds are shown in FIG.
  • the oligonucleotide probe of the present invention can be synthesized by linking the above-mentioned intermediate compound with an oligonucleotide. Introduction of the intermediate compound into the oligonucleotide can be carried out on the automatic DNA synthesizer at the same time as the oligonucleotide synthesis.
  • the oligonucleotide probe of the present invention and the above-mentioned intermediate compound can be synthesized and used in any of D-form and L-form, or may be a mixture thereof.
  • the present invention also relates to a carrier having the oligonucleotide probe immobilized thereon.
  • the carrier is not particularly limited as long as it has a functional group capable of covalently bonding with the reactive functional group of the oligonucleotide probe of the present invention on its surface.
  • the base material of the carrier include glass such as quartz glass, borosilicate glass and soda lime glass, silicon, fiber, wood, paper, ceramics, and plastics (eg, polyester resin, polyethylene resin, polypropylene resin,
  • ABS resin (Acrylonitrile Butadiene Styrene resin), Nylon, Atalinole resin, Fluororesin / Polycarbonate resin, Polyurethane resin, Methylpentene resin, Phenol resin, Melamine resin, Epoxy resin, Vinyl chloride resin).
  • glass, silicon, ceramics, and plastic it is preferable to use glass, silicon, ceramics, and plastic.
  • the oligonucleotide probe of the present invention is immobilized by using a substrate in which a functional group is introduced on the surface of the substrate as a carrier. When the reactive functional group of the oligonucleotide probe is protected, it is preferable to remove the protecting group before immobilization.
  • Examples of the functional group capable of covalently bonding to the reactive functional group of the oligonucleotide probe include, for example, an active ester group, an epoxy group, an amino group, a dye group, a disulfide group, an aldehyde group, a maleimide group, and a carpoimide group.
  • a carrier into which an active ester group, an epoxy group, an aldehyde group, a carbodiimide group, an isothiocyanate group, or an isocyanate group is introduced.
  • a carrier into which a maleimide group or a disulfide group is introduced is preferably used.
  • the shape of the carrier is not particularly limited, and examples thereof include a base, a thread, a sphere, a bead, a polygon, a powder, and a porous form.
  • the base is preferable.
  • the oligonucleotide probe of the present invention can be bound to a label such as biotin and a fluorescent dye.
  • a label such as biotin and a fluorescent dye.
  • the fluorescent dye include CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas Red, TET, TAMRA, FAM, HEX, ROX, GFP, and the like.
  • the oligonucleotide probes of the present invention can also be conjugated to medicaments.
  • the oligonucleotide probe of the present invention has higher binding efficiency to a target nucleic acid than a conventional probe.
  • the oligonucleotide probe of the present invention is easy to synthesize and purify.
  • An intermediate compound (hereinafter, referred to as an amidite compound) for synthesizing the oligonucleotide probe of the present invention was synthesized, introduced into an oligonucleotide, and the ability of the obtained oligonucleotide probe was evaluated.
  • ⁇ 2 is an amidite compound having only an aromatic group without an amino group.
  • DN Introduction of Y2 into an oligonucleotide by an automatic synthesizer, followed by commercial N-monomethoxytrityl-6-amino. An amino group was introduced using a xyl phosphoramidite compound or a trifluoroacetyl-6-aminohexyl phosphoramidite compound (Glen Research), and an amino group and an aromatic group were added to the end of the oligonucleotide.
  • the amidite compound of Y3, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, and Y11 has an amino group in the molecule. Therefore, after introducing the synthetic amidite compound into the oligonucleotide, it is not necessary to introduce an amino group using a separately available amino group-bonded phosphoramidite compound, and the synthesis of the oligonucleotide probe is one step shorter than before. have.
  • ⁇ 3 and ⁇ 5 differ in the structure of the linking site between the amino and aromatic groups, and ⁇ 6 introduces a longer linear linker between the amino and aromatic groups than ⁇ 5 And the distance of the oligonucleotide probe from the surface of the carrier can be maintained.
  • ⁇ 7 is an amidite compound in which the aromatic group of ⁇ 6 is an anthryl group instead of a naphthyl group.
  • Y8 is an amidite compound in which the introduction site of the linear linker of Y5 is different, and an amino group and an aromatic group are close to each other.
  • Oligonucleotide probes having these at the 5 'end were synthesized by a DN ⁇ automatic synthesizer (X3-Sp, X5-Sp, X6-Sp, X7-Sp, X8-Sp, respectively). , X 9 -S p; FIG. 8).
  • oligonucleotide probe having no aromatic group As the oligonucleotide probe having no aromatic group, the above-mentioned commercially available amino group-bonded phosphoramidite compound was used, and an amino group was introduced at the end of the oligonucleotide (X1-Sp; FIG. 8). An oligonucleotide probe having no aromatic group between the two (X4_Sp; FIG. 8) was used as a comparative example.
  • the synthesized oligonucleotide was purified to a high degree of purity using a reversed-phase column, dissolved in a spot solution for the preparation of an oligo chip at a certain concentration, spotted on a glass substrate, and immobilized.
  • oligo probes (X 2—Sp, X 3—Sp, X 5—Sp, X 6—Sp, X 8 -S p) was found to be highly immobilized (Fig. 9a).
  • the reaction was performed with fluorescein isothiocyanate and Cy 3 succinimidyl ester.
  • the oligonucleotide probe having an aromatic group of the present invention exhibits higher reactivity with any of the fluorescent dyes than the conventional oligonucleotide probe, and improves the reactivity with a chemical substance even in a solution. (Fig. 9 b, c;).
  • the Y9 amidite compound is a derivative that can be introduced into the end of the oligonucleotide or into the chain (FIG. 7). Oligonucleotides in which the Y9 amidite compound was introduced at the terminal (X9-Sp25) and in the chain (X9-Sp35) were synthesized. As with other amidites, their reactivity with fluorescent dyes (Fig. 9c) and immobilization efficiency on glass substrates (Fig. 12) were examined.
  • the extract was washed once with 100 ml of a saturated saline solution, and the organic layer was dried over sodium sulfate. After concentrating the solution under reduced pressure, the obtained oily substance was dissolved in 80 ml of dimethylformamide under an argon atmosphere, and 1.6 g (3 equivalents) of sodium azide and 1.75 g of ammonium chloride (3 g) were added. (4 equivalents) was added and the mixture was heated and stirred at 80 ° C for 2 hours.
  • N-trifluoroacetyl-6-aminocaproic acid (disulfide compound f) 360 mg (1.3 equivalents) and 1,1'-carboerdiimidazole 23.5 mg (1.2 was dissolved in 1 ml of dimethylformamide and stirred at room temperature for 2 hours.
  • the obtained oily substance was dissolved in 75 ml of methylene chloride, cooled to 0 ° C, and 2.1 ml of trifluoroacetic acid (90% aqueous solution) was added. The mixture was stirred at C for 1 hour. To the reaction solution was added 80 ml of a saturated aqueous solution of sodium hydrogencarbonate, and the mixture was returned to room temperature. The organic layer was washed once with 8 Om 1 of a saturated aqueous solution of sodium hydrogencarbonate and once with 8 Om 1 of a saturated saline solution, and dried over sodium sulfate.
  • reaction solution was concentrated under reduced pressure, acetic acid was removed by azeotropy with toluene, and the residue was purified by silica gel column chromatography (eluting solvent: ethanol-chloroform) to give 1.4 lg (yield) of the title compound (compound n). 83%) as a pale yellow solid.
  • the extract was washed once with 1 and once with 100 ml of a saturated saline solution, and the organic layer was dried over sodium sulfate. After the solution was concentrated under reduced pressure, the obtained oily substance was dissolved by adding 70 ml of an 80% aqueous acetic acid solution, followed by stirring at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, acetic acid was removed by azeotrope with toluene, and azeotrope with pyridine. The residue was dissolved in 60 ml of pyridine under an argon atmosphere, and 4.07 g (1.2 equivalents) of dimethoxytritin chloride was added, followed by stirring at room temperature for 2 hours.
  • Oligonucleotide synthesis was performed on an Applied Biostems type 394 DNA / RNA synthesizer.
  • the analysis was performed using a Waters 996 photodiode array detector using a Gison son apparatus for HP LC.
  • Waters ⁇ Bondasphere C 183 00 A (internal diameter 3.9 mm x length 150 mm) as a column for reversed-phase analysis
  • GL Science Inertsi as a column for reversed-phase separation 1 ODS-3C18 (8.0 mm ID x 300 mm length)
  • Tosoh T SK—GE LDE AE—2 SW (4.6 mm ID ⁇ 250 mm length) for anion exchange analysis used.
  • TEAA triethylammonium acetate buffer
  • anion exchange concentration gradient of ammonium formate in 20% acetonitrile in water '.
  • oligonucleotide probes were prepared from doxynucleoside 3'-phosphoramidite (purchased from Nippon Techno Service Co., Ltd.) and used as an automatic DNA synthesizer (Model 394A; PerkinElmer Japan, Inc. 'Applied Dubai) (Manufactured by Systems Division) at 0.2 or 1 ⁇ mo 1 scale.
  • Cy5-AS-Sp 5'-Cy5-ATCGTCATCATCGTCGTC CATGTGGTCATGGCAAACATTGGAATTGCTTGGAA GAGTTTC—3 '(SEQ ID NO: 2)
  • N-monomethoxytrityl-6-aminohexyl phosphoramidite (Glen Research) was used for the amino group introduced at the 5 'end of the X1-Sp and X4-Sp oligonucleotides.
  • the introduction of the amino group of Y 2, ⁇ 3, ⁇ 5 , ⁇ 6, ⁇ 7, and ⁇ 8 were performed using phosphoramidite compounds.
  • FIG. 8 shows the structures of the synthesized oligonucleotide probes X 1 — Sp to X 8 — Sp.
  • the oligonucleotide probe was treated and purified as follows.
  • the oligonucleotide probe was cut out from CPG (Controlled Glass) with concentrated aqueous ammonia, and then 50. Heated at C for 12 hours. After distilling off the solvent and dissolving in deionized water, C18 (manufactured by Waters) open force column chromatography was performed (column size 0.8 x 18 cm: 5-50% acetate etol. 1M triethylammonium acetate (hereinafter referred to as “TEAA”) eluted with a linear concentration gradient using an aqueous solvent.
  • CPG Controlled Glass
  • C18 manufactured by Waters
  • TEAA triethylammonium acetate
  • the conditions for reversed phase HP LC were as follows:
  • 1-3-3- is an oligonucleotide complementary to X 1 —Sp.
  • X 1 —Sp oligonucleotide complementary to X 1 —Sp.
  • Oligonucleotide probe (XI-AS-Sp) (1 nmo1) and Cy5-succinimidyl ester (Pharmacia) (500 nmol) were added to 10% (v / v) dimethylformamide, 0% The solution was dissolved in a 25 M carbonate buffer solution (total volume: 100 L), and the reaction was carried out at 35 ° C under light shielding. Sixteen hours after the start of the reaction, desalting was performed with NAP10 (Pharmacia). Thereafter, fractionation was performed with reversed-phase HPLC. Column: ⁇ —bonder sphere (C-18) column ⁇ 3.9 x 150 mm ( ⁇ made by Otters)
  • Slide glass (20 pieces) was immersed in a 10% aqueous sodium hydroxide solution (200 mL) for 15 minutes, and then water (twice with 200 mL) and a 1% aqueous hydrochloric acid solution (200 mL) were used. L) followed by water (twice with 200 mL). It was immersed in methanol (200 mL), subjected to ultrasonic cleaning for 5 minutes, dried by centrifugation, and further dried at 180 degrees for 3 hours.
  • the dried slide glass was immersed in 3-aminotrimethoxysilane (13 mL), water (8 mL), and methanol (380 mL), and stirred at room temperature for at least 5 hours. Thereafter, the slide glass was taken out, washed three times with methanol (200 mL), centrifuged, and dried at 180 degrees for 3 hours.
  • 1,4-phenylenediisothiocyanate (1400 mg) was dissolved in a 10% pyridine.dimethylformamide solution (220 mL), and the slide glass which had been subjected to the above aminosilane diluting was dissolved in the solution. The mixture was stirred at room temperature for 16 hours.
  • an isothiosinate slide glass is spread on a glass container, and a 20 or 30% tris (3-aminopropyl) amine (methanol solution (130 ⁇ L)) is added dropwise thereto, and the mixture is sealed and sealed at 37 ° C. For 5 hours. Thereafter, the slide glass was washed twice with methanol (200 mL) and once with acetone (200 mL). After drying at room temperature under reduced pressure for 1 hour, 1,4-phenylene diisothiocyanate (1400 mg) was dissolved in 10% pyridine 'dimethylformamide solution (220 mL) in advance. The slide glass was put therein and stirred at room temperature for 16 hours.
  • a 50-base oligonucleotide probe (Xn-Sp) (50-300 pmo1) is dissolved in sterile water (51), and a spot solution (5 ⁇ l; 1M carbonate buffer ( ⁇ 9.0)) is dissolved. And spotted on a coated slide glass coated with 1,4-phenylenediisocarbonate by a spotter (SPBI ⁇ 2000, manufactured by Hitachi Software Engineering Co., Ltd.). After spotting, filter paper was spread on a tight box, and a 300 mM aqueous solution of sodium hydrogen phosphate was wetted. A slide glass that had been spotted was added to prevent the solution from sticking.
  • the glass was immersed in a 1M aqueous solution of ethanolamine (20 OmL), and stirred at room temperature for 1 hour to perform blocking. After washing three times with sterile water, dry in the draft It was dried and refrigerated.
  • the oligonucleotide probe of the present invention can be detected with higher sensitivity than X 1 —Sp or X 4 —Sp.
  • the glass was allowed to stand at room temperature. After 16 hours, remove the slide glass from the tight box, and remove the slide glass with 0.1% Triton X (200 mL) for 5 minutes at room temperature, and with a 2% aqueous hydrochloric acid solution (200 mL) for 2 minutes at room temperature. The plate was washed with 1 M lithium chloride (200 mL) for 10 minutes at room temperature and with sterile water (200 mL) for 1 minute at room temperature. Subsequently, the glass was immersed in a 1 M aqueous ethanolamine solution (200 mL), and stirred at room temperature for 1 hour to perform blocking. After washing three times with sterile water, the mixture was dried in a draft and stored refrigerated.
  • X 10 -Sp 25 has an amino group protected by a monomethoxytrityl group (MMT r).
  • MMT r monomethoxytrityl group
  • a 10% aqueous solution of acetic acid (I mL) was added to the oligonucleotide, and the mixture was treated at room temperature for 5 minutes.
  • the reaction solution was concentrated under reduced pressure, and water was added, and the mixture was azeotroped under reduced pressure. After three azeotropes, the residue was dissolved in 1 mL of sterile water and analyzed by reversed-phase HPLC (Fig. 14b, c). From the above, it can be seen that X 10 — Sp 25 was deprotected under mildly acidic conditions, resulting in the same structure as X 8 — Sp 25 (Fig. 14a).
  • the present invention makes it possible to reduce the cost of DNA chips, and is expected to contribute to the widespread use of DNA chips as a means of gene diagnosis technology. Sequence listing free text

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Abstract

Une sonde oligonucléotidique solidement fixée à un support et qui maintient efficacement un acide nucléique cible complémentaire. Il s’agit d’une sonde oligonucléotidique représentée par la formule générale B-D-A (où A représente un oligonucléotide ; D représente un groupe organique divalent ayant au moins un groupe aromatique ; et B représente un groupe fonctionnel réactif ou une forme protégée de celui-ci.)
PCT/JP2005/007666 2004-04-20 2005-04-15 Sonde oligonucléotidique Ceased WO2005103247A1 (fr)

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WO2007013190A1 (fr) * 2005-07-27 2007-02-01 National Institute Of Advanced Industrial Science And Technology Sonde oligonucléotidique
WO2013024694A1 (fr) * 2011-08-12 2013-02-21 独立行政法人産業技術総合研究所 Vecteur en phase solide pour un oligonucléotide aminé
WO2018013999A1 (fr) 2016-07-15 2018-01-18 Am Chemicals Llc Supports solides non nucléosides et blocs de construction de phosphoramidite pour synthèse d'oligonucléotides

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JP6249436B2 (ja) * 2012-08-31 2017-12-20 国立大学法人東京農工大学 四重らせんｄｎａ検出プローブ及びそれを用いた四重らせん構造の検出方法

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WO2007013190A1 (fr) * 2005-07-27 2007-02-01 National Institute Of Advanced Industrial Science And Technology Sonde oligonucléotidique
US7491857B2 (en) 2005-07-27 2009-02-17 National Institute Of Advanced Industrial Science And Technology Oligonucleotide probe
WO2013024694A1 (fr) * 2011-08-12 2013-02-21 独立行政法人産業技術総合研究所 Vecteur en phase solide pour un oligonucléotide aminé
JPWO2013024694A1 (ja) * 2011-08-12 2015-03-05 独立行政法人産業技術総合研究所 アミノ化オリゴヌクレオチド用固相担体
WO2018013999A1 (fr) 2016-07-15 2018-01-18 Am Chemicals Llc Supports solides non nucléosides et blocs de construction de phosphoramidite pour synthèse d'oligonucléotides
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates

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