EP2971086A1 - Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation - Google Patents

Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation

Info

Publication number
EP2971086A1
EP2971086A1 EP14714494.3A EP14714494A EP2971086A1 EP 2971086 A1 EP2971086 A1 EP 2971086A1 EP 14714494 A EP14714494 A EP 14714494A EP 2971086 A1 EP2971086 A1 EP 2971086A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
pyrazolo
pyrimidin
alkynyl
analogue
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.)
Withdrawn
Application number
EP14714494.3A
Other languages
German (de)
English (en)
Inventor
Alexei Vorobiev
Eugeny A. Lukhtanov
Noah Scarr
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.)
Elitechgroup BV
Original Assignee
Elitechgroup BV
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
Priority claimed from US13/827,456 external-priority patent/US8969003B2/en
Application filed by Elitechgroup BV filed Critical Elitechgroup BV
Publication of EP2971086A1 publication Critical patent/EP2971086A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/101—Modifications characterised by incorporating non-naturally occurring nucleotides, e.g. inosine

Definitions

  • This invention relates to universal bases and their uses.
  • Universal bases are extensively used in primers, probes, hybridization, sequencing, cloning and the diagnostic detection of infectious targets.
  • a universal base analogue forms base pairs with each of the natural bases with little discrimination between them (Loakes et al., 1997; Loakes, 2001 ).
  • Desirable requirements for a universal base include the ability to: a) pair with all natural bases equally in a duplex, b) form a duplex which primes DNA synthesis by a polymerase, c) direct incorporation of the 5 '-triphosphate of each of the natural nucleosides opposite it when copied by a polymerase, (d) be a substrate for polymerases as the 5 '-triphosphate, e) be recognized by intracellular enzymes such that DNA containing them may be cloned. (Loakes et al., 1997). At present no analogue has been shown to have all these characteristics.
  • a universal 2 '-deoxy inosine analogue 7-octadiynyl-7-deaza-2'-deoxyinosine has also been disclosed (Ming et al., 2008).
  • the nucleobase of this analogue shows universal binding properties with the four natural bases in a 12-mer oligonucleotide with T m 's that varies from 45°C for C to 34°C for G.
  • the present disclosure pertains to functional i/ed 3-alkynyl- 1 H- pyrazolo[ 3.4-d )pyrimidin-4(5H)-ones as universal bases and their methods of use.
  • 3-alkynyl- lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one-based analogues function unexpectedly well as universal bases. Not only do they stabilize duplexes substantially more than hypoxanthine opposite A, C. and T but they are also recognized in primers by polymerases, allowing efficient amplification.
  • l H-Pyrazolo[3,4-d]pyrimidin- 4(5H)-ones substituted at the 3-position with hydroxylalkynyl (IPPOH) or aminoalkynyl (IPPNH 2 ) are preferred as universal bases.
  • the 3-alkynyl-lH-pyrazoIo[3,4-d]pyrimidin-4(5H)-one analogues can be incorporated into novel nucleic acid primers and probes. They do not significantly destabilize nucleic acid duplexes, as other universal bases do. As a result, the novel nucleic acid primers and probes incorporating the 3-alkynyl-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues can be used in a variety of methods.
  • 3-alkynyl- 1 H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues can also be substituted with pyrene or acridine to further increase duplex stability. It is to be appreciated that other similar polyaromatics with 0 to 3 hetero atoms will produce similar stabilization.
  • Figure 1 shows the structure of 2 ' -deoxyinosine and 2-deoxy-p-D- ribofuranosyl- (5H)-ones.
  • Figure 2 shows the possible hydrogen bonds between hypoxanthine and the natural nucleic acid bases.
  • Figure 3 shows, without being bound by theory, proposed hydrogen bonds between 3-(aminobutynyl)-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one and the normal nucleic bases.
  • Figure 4 shows a reaction scheme for synthesis of a protected (2-deoxy-p- D-ribofuranosy l)-3 -hydroxybuyny 1 - 1 H-pyrazolo[3 ,4-d]pyrimidin-4(5 H)-one 5 ' - phosphoramidite.
  • Figure 5 shows a reaction scheme for synthesis of a protected (2-deoxy-P- D-ribofuranosyl)-3-hydroxybuynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 3'- phosphoramidite.
  • Figure 6 shows a reaction scheme for synthesis of a protected inosine 5'- phosphoramidite.
  • Figure 7 shows a reaction scheme for synthesis of protected (2-deoxy- -D- ribofuranosyl)-3-(aminoalkynyl)-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues.
  • Figure 8 shows a reaction scheme for synthesis of (2-deoxy-P-D- ribofuranosyl)-3-(methylcarbamoyloalkynyl)-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues.
  • Figure 9 shows a reaction scheme for synthesis of (2-deoxy- -D- ribofuranosyl)- 1 H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues bearing guanidinoalkynyl substituates at the 3-position of the nucleobase.
  • Figure 10 shows a summary of melting temperatures (T m s) of duplexes between the 15-mer GTAAGXAGXCATAAC (SEQ ID NO: 1), where X is independently 2'- deoxinosine or a 2-deoxy-p-D-ribofuranosyl- 3-alkynyl- 1 H-pyrazolo[3,4-d]pyrimidin-4(5H)- one of the present disclosure, and the complement which contains either A, T, C or G opposite to X.
  • T m s melting temperatures
  • Figure 1 1 shows a comparison of T m s of duplexes between the 1 5-mer GTAAGXAGACATAAC (SEQ ID NO:2), where X is independently 2'-deoxinosine or a 2- deoxy- ⁇ - D-r i bo furanosy 1 - 3-alkynyl-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one of the present disclosure, and the complement which contains either A, T, C or G opposite to X.
  • Figure 12 shows a comparison of T m s of duplexes between the 15-mer GTAAGTAGXCATAAC (SEQ ID NO:3), where X is independently 2 '-deoxinosine or a 2- deoxy- -D-ribofuranosyl- 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one of the present disclosure, and the complement which contains either A, T, C or G opposite to X.
  • X is independently 2 '-deoxinosine or a 2- deoxy- -D-ribofuranosyl- 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one of the present disclosure, and the complement which contains either A, T, C or G opposite to X.
  • Figure 13 shows a comparison of T m s of duplexes between the 15-mer GTAAGXAGXCATAAC (SEQ ID NO: 1 ), where X is independently 2'-deoxinosine, or a 2- deoxy- -D-ribofuranosyl- 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one of the present disclosure, and the complement which contains either A, T, C or G opposite to X.
  • Figure 14 shows a comparison of melting and real-time PGR data for Adenovirus assays using primers containing the currently described nucleoside analogues.
  • Figure 15 shows a comparison of melting and real-time PCR data for Adenovirus assays using primers containing multiple incorporations of the currently described nucleoside analogues.
  • Figure 16 shows a comparison of Cts for a Meticillin-resistant Staphylococcus aureus LGA251 target assay using primers substituted with five deoxyinosine or five 3-(aminobutynyl)- l h-pyra/olo
  • Figure 17 shows that when two Ts in a primer are substituted with either deoxyinosine or 3-(aminobutynyl)- l h-pyrazolo[3,4-d]pyrimidin-4(5h)-one nucleotides that the polymerase incorporate two Cs complementary to the Ts.
  • Figure 18 shows a reaction scheme for synthesis of 3-aminoalkynyl- substituted (2-deoxy-p-D-ribofuranosyl)-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one 5'- phosphoramidite 35.
  • Figure 19 shows a reaction scheme for post-synthetic conjugation of pyrene and acridine carboxylic acids with 3-(aminobutynyl)-l H-pyrazolo[3,4-d]pyrimidin-4(5H)- one.
  • Figure 20 shows a comparison of melting temperatures of DNA duplexes containing 107, 107-Pyr, I07-(Ac-Pyr)i, I07-(Ac-Pyr) 2 , 107-Bu-Pyr base analogues, paired with all four natural bases.
  • Figure 21 shows a comparison of T m s of duplexes between the 15-mer GTAAGXAGACATAAC, where X is independently deoxyinosine, 104, 107, 107-Pyr, 107- Acr, and the complement which contains either A, T, C or G opposite to X.
  • Figure 22 shows a comparison of T m s of duplexes between the 15-mer GTAAGTAGXCATAAC, where X is independently deoxyinosine, 104, 107, 107-Pyr, 107- Acr, and the complement which contains either A, T, C or G opposite to X,
  • Figure 23 shows a comparison of T m s of duplexes between the 15-mer GTAAGXAGXCATAAC, where X is independently deoxyinosine, 104, 107, 107-Pyr, 107- Acr, and the complement which contains either A, T, C or G opposite to X.
  • target sequence refers to a sequence in a target RNA, or DNA that is partially or fully complementary to the mature strand.
  • the target sequence can be described using the four bases of DNA (A, T, G, and C), or the four bases of RNA (A, Li, G, and C).
  • Complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes, including the wobble base pair formed between U and G. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine.
  • substantially complementary refers to the ability of an oligonucleotide to form base pairs specifically with another oligonucleotide where said oligonucleotide may contain one or more mismatches.
  • duplex refers to a double stranded structure formed by two complementary or substantially complementary polynucleotides that form base pairs with one another, including Watson-Crick base pairs and U-G wobble pairs that allow for a stabilized double stranded structure between polynucleotide strands that are at least partially complementary.
  • the strands of a duplex need not be perfectly complementary for a duplex to form, i.e.. a duplex may include one or more base mismatches.
  • duplexes can be formed between two complementary regions within a single strand (e.g., a hairpin).
  • nucleotide refers to a ribonucleotide or a
  • Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogues.
  • purines e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues
  • pyrimidines e.g., cytosine, uracil, thymine, and their derivatives and analogues.
  • Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR. Ni l;. NHR, NR2, or CN, wherein R is an alkyl moiety.
  • Nucleotide analogues are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2'-methyl ribose, non-natural phosphodiester linkages such as in ethylphosphonates, phosphorothioates and peptides.
  • modified bases refers to those bases that differ from the naturally-occurring bases (adenine, cytosine, guanine, thymine, and urasil) by addition or deletion of one or more functional groups, differences in the heterocyclic ring structure (i.e., substitution of carbon for a heteroatom, or vice versa), and/or attachment of one or more linker arm structures to the base.
  • Preferred modified nucleotides are those based on a pyrimidine structure or a purine structure, with the latter more preferably being 7 deazapurines and their derivatives and pyrazolopyrimidines (described in PCT WO 01/84958); and also described in U.S. Patent No. 6, 127, 121.
  • Preferred modified bases are 5- substituted pyrimidines and 3-substituted pyrazolopyrimidines.
  • Examples of preferred modified bases are 6-amino- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (PPG or Super G ), 4-am i no- 1 H-py razo lo[ 3.4-d ] p rim id ⁇ ne. l//-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7/ )-dione. 6- amino-3-prop- 1 -ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one,
  • universal bases and “degenerative bases” refer to natural base analogues that are capable of forming base pairs with two or more natural bases in DNA or RNA with little discrimination between them. Universal and degenerative bases are well known in the art and disclosed in U.S. Patent No. 7,348,146 that is incorporated by reference. Oligonucleotide conjugates containing an inosine analog of the current disclosure may also comprise one or more universal and degenerative bases, in addition to the naturally-occurring bases adenine, cytosine, guanine, thymine and uracil.
  • nucleotide is also meant to include what are known in the art as universal bases.
  • universal bases include, but are not limited to, 3- nitropyrrole, 5-nitroindole, or nebularine.
  • nucleotide is also meant to include the N3' to P5' phosphoramidate, resulting from the substitution of a ribosyl 3 '-oxygen with an amine group.
  • nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
  • linker refers to a moiety that is used to assemble various portions of the molecule or to covalently attach the molecule (or portions thereof) to a solid support. Additionally, a linker can include linear or acyclic portions, cyclic portions, aromatic rings or combinations thereof.
  • protecting group refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Greene and P.O. Puts. Protective Groups in Organic Chemistry. (Wiley, 2nd cd.
  • Representative amino protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), fcr/-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC) and the like.
  • hydroxy protecting groups include those where the hydroxy group is either acylated or alkylated such as benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. These protecting groups can be removed under conditions which are compatible with the integrity of a compound of interest. Deprotection conditions are well known in the art and described in the references above.
  • alkyl refers to a linear, branched, or cyclic saturated monovalent hydrocarbon radical or a combination of cyclic and linear or branched saturated monovalent hydrocarbon radicals having the number of carbon atoms indicated in the prefix.
  • (Ci-C 8 )alkyl is meant to include methyl, ethyl, «-propyl, 2-propyl, tert-butyl, pentyl, cyclopentyl, cyclopropylmethyl and the like.
  • quenchers are described in co-owned U.S. Patent No. 6,727,356, incorporated herein by reference.
  • Other quenchers include bis azo quenchers (U.S. Patent No. 6,790,945) and dyes from Biosearch Technologies, Inc. (provided as Black HoleTM Quenchers: BH-1 , BH-2 and BH-3), Dabcyl. TAMRA and carboxytetramethyl rhodamine.
  • Minor groove binder oligonucleotide conjugates have been described (see U.S. Patent No. 5,801 , 155 and U.S. Patent No. 6,312,894, both hereby incorporated by reference). These conjugates form hyper-stabilized duplexes with complementary DNA .
  • sequence specificity of short minor groove binder probes is excellent for high temperature applications such as PG R.
  • the probes/conjugates of the present disclosure can also have a covalcntly attached minor groove binder.
  • suitable minor groove binders have been described in the literature. See, for example, Kutyavin. et al. U.S. Patent No.
  • Suitable methods for attaching minor groove binders (as well as reporter groups such as fluorophores and quenchers) through linkers to oligonucleotides are described in, for example, U.S. Patent Nos. RE 38,416; 5,512,677; 5,419,966; 5,696,251 ; 5,585,481 ; 5,942,610 and 5,736,626.
  • a nucleotide mono-phosphate, nucleotide di-phosphate or a nucleotide triphosphate processing enzyme is an enzyme that utilizes a nucleotide mono-phosphate, nucleotide di-phosphate or a nucleotide triphosphate as one of its substrates.
  • a nucleotide mono-phosphate, a nucleotide di-phosphate or a nucleotide triphosphate nucleic acid processing enzyme catalyzes modifications to nucleic acids or nucleic acid intermediates using either a nucleotide mono-phosphate, nucleotide di-phosphate or a nucleotide triphosphate as one of the substrates.
  • Nucleotide mono-phosphate, nucleotide di-phosphate or nucleotide triphosphate enzymes include but are not limited to primer extension enzymes, DNA polymerases, RNA polymerases, restriction enzymes, nicking enzymes, repair enzymes or ligation enzymes.
  • Amplification procedures are those in which many copies of a target nucleic acid sequence are generated, usually in an exponential fashion, by sequential polymerization and/or ligation reactions.
  • the present invention is useful in ampl ifications involving three- way j unctures (see, WO 99/37085), signal amplification (see Capaldi, et al., Nuc. Acids Res. , 28:E21 (2000)), T7 polymerases, reverse transcriptase, RNase H, R I -PCR. Rolling Circles, cleavase and the like. Isothermal amplification methods have been reviewed (cc Niemz, A.
  • the "term oligonucleotide primers adjacent to a probe region” refers to when 0 or one or more base separate the primer and probe.
  • the term “overlapping with said probe region” is defined as disclosed in U.S. Patent No. 7,319,022.
  • the term “Ct” refers to the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold.
  • a primer is a nucleic acid that is capable of hybridizing to a second, template nucleic acid and that, once hybridized, is capable of being extended by a polymerizing enzyme (in the presence of nucleotide substrates), using the second nucleic acid as a template.
  • Polymerizing enzymes include, but are not limited to, DNA and RNA polymerases and reverse transcriptases, etc. Conditions favorable for polymerization by different polymerizing enzymes are well-known to those of skill in the art. See, for example, Sambrook et al, supra; Ausubel, et al, supra; Innis et al., supra.
  • a primer in order to be extendible by a polymerizing enzyme, a primer must have an unblocked 3 '-end, preferably a free 3 ' hydroxyl group.
  • the product of an amplification reaction is an extended primer, wherein the primer has been extended by a polymerizing enzyme.
  • the present inosine analogues include monomeric compounds of Formula I and II:
  • R is H or a protecting group
  • R 2 is H, a phosphate group, a polyphosphate group, an activated phosphate group, a protecting group, phosphoramidite or a solid support;
  • R 3 is H, a protecting group, or a phosphoramidite
  • R 4 is I f or an alkyl
  • R " is pyrene or acridine
  • n 1 to 5;
  • y is 1 to 10.
  • this resulting polyphosphate analog can be a diphosphate or triphosphate.
  • These polyphosphate analogs can be used in enzyme catalyzed primer extension reactions.
  • the present inosine analogues are also useful in oligomers and in intermediates for oligonucleotide synthesis.
  • the inosine analogues can also include compounds of Formulas III and IV below:
  • R 1 is H
  • L is a sugar or sugar/phosphate backbone analogue, including but not limited to a backbone of DNA, RNA, PNA, locked nucleic acid, modified DNA, modified PNA, modified RNA. or any combination thereof;
  • R 6 is H or alkyl
  • n 1 to 5.
  • the modified oligonucleotides incorporating the present inosine analogues are comprised of glycosidic moieties, preferably 2- deoxyribofuranosides wherein all internucleoside linkages are the naturally occurring phosphodiester linkages.
  • the 2-deoxy-P-D-ribofuranose groups are replaced with other sugars, for example, ⁇ -D-ribofuranose.
  • ⁇ -D- ribofuranose may be present wherein the 2-OH of the ribose moiety is alkylated with a C 1 -5 alkyl group (2-(0— C alkyl) ribose) or with a C2-6 alkenyl group (2-(0— C2-6 alkenyl) ribosc). or is replaced by a fluoro group (2-fluororibose).
  • Related oligomer-forming sugars useful in the present invention are those that are "locked' * , i.e.. contain a methylene bridge between C- 4 * and an oxygen atom at C-2 " .
  • oligonucleotide can also be used, and arc known to those of skill in the art, including, but not limited to, a-D-arabinofuranosides, a-2'-deoxyribofuranosides or 2'.3'-dideo ⁇ y-3'- aminoribofuranosides.
  • Oligonucleotides containing a -D-arabinofuranosides can be prepared as described in U.S. Patent No. 5, 177, 196.
  • Oligonucleotides containing 2',3'-dideoxy-3'- aminoribofuranosides are described in Chen et al. 1995.
  • oligonucleotides containing 2'-halogen- 2'-deoxyribofuranosides have been described.
  • the phosphate backbone of the modified oligonucleotides described herein can also be modified so that the oligonucleotides contain phosphorothioate linkages and/or methvlphosphonates and/or phosphoroamidates (Chen et al., 1995). Combinations of oligonucleotide linkages are also within the scope of the present invention. Still other backbone modifications are known to those of skill in the art.
  • the inosine analogues described herein are incorporated into PNA and DNA/PNA chimeras to balance T m s and provide modified oligonucleotides having improved hybridization properties.
  • Various modified forms of DNA and DNA analogues have been used in attempts to overcome some of the disadvantages of the use of DNA molecules as probes and primers.
  • PNAs peptide nucleic acids
  • PNAs also known as polyamide nucleic acids (Nielsen et al. 1991 ).
  • PNAs contain natural RNA and DNA heterocyclic base units that are linked by a polyamide backbone instead of the sugar-phosphate backbone characteristic of DNA and RNA.
  • PNAs are capable of hybridization to complementary DNA and RNA target sequences and, in fact hybridize more strongly than a corresponding nucleic acid probe.
  • the synthesis of PNA oligomers and reactive monomers used in the synthesis of PNA oligomers have been described in U.S. Patent Nos. 5,539,082; 5,714,331 ; 5,773,571 ; 5,736,336 and 5,766,855.
  • Alternate approaches to PNA and DNA/PNA chimera synthesis and monomers for PNA synthesis have been summarized (Uhlmann et al. 1998). Accordingly, the use of any combination of normal bases.
  • Example compounds of the invention are shown in Figure 1 .
  • Figure 2 illustrates the hydrogen bonds that occur between hypoxathine and the natural nucleic acid basis. As indicated, and without being bound by theory, those skilled in the art view hypoxanthine as forming two hydrogen bonds with the normal nucleic acid bases in duplex formation.
  • Figure 3 illustrates, again without being bound by theory, proposed hydrogen bond formation with NH 2 Bu-PPI with natural bases in a duplex.
  • the present 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one-based analogues function unexpectedly well as universal bases. Not only do they stabilize duplexes substantially more than hypoxanthine opposite A, C, and T but they are also recognized in primers by polymerases, allowing efficient amplification. In the case of G, binding is similar to that observed with inosine.
  • the 3-alkynyl-l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one-based analogues are further substituted with pyrene or acridine to provide increased duplex stability.
  • nucleic acid processing enzyme concerns any enzyme that is involved in a chemical transformation or physical manipulation of nucleic acids or their components.
  • 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)- one-based analogues are useful in all hybridization based techniques, including but not limited to detection of more than one target, amplification of more than one target, use of arrays, use of processing enzymes, conversion of intermediates, sequencing, and others.
  • the present disclosure pertains, in one aspect, to a method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence the method comprising:
  • nucleic acid polymer has a backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and at least one nucleic acid base is substituted with a 3- alkynyl-1 H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture;
  • At least one of said oligonucleotide primers has a sequence complementary to an adjacent portion of the probe region of the target nucleic acid sequence.
  • the method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence includes methods in which each base independently represents a nucleic acid base, at least one 3-alkynyl-l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one analogue, and at least one modified base.
  • nucleic acid oligomer is a conjugate comprising a minor groove binder ligand.
  • Another embodiment pertains to a method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence, the method comprising one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, wherein said one or more oligonucleotide primers is an oligonucleotide is between 5 and 50 bases long wherein said nucleic acid polymer has a backbone component selected from the group consisting of a sugar phosphate backbone, a chimeric modified sugar phosphate backbone, chimeric locked nucleic acid backbone, a chimeric peptidic backbone or a variant thereof used in nucleic acid amplification; and at least one nucleic acid base is substituted with a 3-alkynyl- l H-pyrazoIo[3,4-d]pyrimidin-4(5H)-one analogue and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amp
  • Another method for primer extension of nucleic acids targets comprises one or more primers complementary to the target sequence, wherein each nucleic acid base independently represents a nucleic acid base, at least one 3-alkynyl- 1 1 l-pyrazolo[3.4- d]pyrimidin-4(5H)-one analogue and at least one modified base
  • the primer also contains a minor groove binder ligand and a label.
  • An alternative method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence comprises one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, wherein each nucleic acid base independently represents a nucleic acid base, at least one 3-alkynyl- 1 H- pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue and at least one modified base.
  • multiple nucleic acid targets are detected in a polymerase amplification reaction with one or more primers and more than one probe where each such probe is uniquely labeled and wherein at least one of said primers or probe contains at least one normal base substituted with a 3-alkynyl-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue.
  • the present disclosure pertains to a method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, the method comprising: a) measuring the fluorescence emission as a function of temperature to determine a first melting profile of a first probe melting from a first amplified polynucleotide and a second melting profile of a second probe melting from a second amplified polynucleotide wherein each probe independently contains zero or one or more 3-alkynyl- l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one analogues; and
  • the sample is further contacted with a set of primers under amplification conditions and where at least one of the primers contains at least one 3- alkynyl-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues.
  • at least one f the primers may also contain one modified base selected from the group disclosed above.
  • oligonucleotide conjugate comprising a fluorophore
  • the oligonucleotide conjugate has a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, and a peptidic backbone
  • oligonucleotide conjugate contains a nucleic acid base substituted with a 3-alkynyl-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue at the site complementary to said second single nucleotide polymorphism, and
  • oligonucleotide conjugate has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture
  • the polymerization is catalyzed by a polymerizing enzyme under isothermal conditions.
  • a nucleotide comprising a 3-alkynyl-l H- pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue as disclosed herein is incorporated by a nucleotide processing enzyme into a nucleic acid, where said nucleic acid as a result has improved hybridization properties when hybridized to a second nucleic acid.
  • Incorporated 3- alkynyl-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues of the invention have universal properties including improved hybridization (T m s) particularly with A, C and T. While the present analogues will hybridize to G, they typically show no improvement i hybridization compared to inosine.
  • modified oligomers comprising 3- alkynyl-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues as disclosed herein, are useful in techniques including, but not limited to, hybridization, primer extension, hydrolyzable probe assays, amplification methods (e.g., PCR, SSSR, NASBA, SDA, LAMP ), single nucleotide mismatch discrimination, allele-specific oligonucleotide hybridization, nucleotide sequence analysis, hybridization to oligonucleotide arrays, in itu hybridization and related techniques.
  • amplification methods e.g., PCR, SSSR, NASBA, SDA, LAMP
  • Oligomers disclosed herein can be used as immobilized oligomers in oligomer arrays such as those described in, for example, U.S. Patent Nos. 5,492,806; 5,525,464; 5,556,752 and PCT publications WO 92/10588 and WO 96/17957.
  • sequencing primers contain one or more 3-alkynyl- l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue bases.
  • kits that contain probes and/or conjugates as described above, along with primers for amplification reactions, wherein the primers contain one or more 3-alkynyl-l H-pyrazoIo[3,4-d]pyrimidin- 4(5H)-one analogue bases, more preferably, from one to ten 3-alkynyl- l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one bases per primer.
  • the protected phosphoramidite 8 was synthesized by reaction of 7 with 2-cyano-N,N,N ' ,N ' -tetraisopropy Iphord iamidite and diisopropylammonium tetrazolide in Cl ⁇ Ck
  • the protected phosphoramidite 12 was synthesized by reaction of 11 with 2-cyano-N,N,N',N'- tetraisopropylphordiamidite and diisopropylammonium tetrazolide in CH2CI2.
  • Amines 25, 26 and 27 were prepared from the protected amine intermediates 19, 21 and 23 by a treatment with a mixture of aqueous methylamine and concentrated ammonium hydroxide at 55°C under pressure.
  • the free amines were converted into methylcarbomoyl derivatives 28, 29 and 30 by a reaction with N-succinimidyl N- methylcarbomate.
  • the resulting 3 '-hydroxy intermediates were reaction with 2-cyanoethyl tetraisopropylphosphordiamidite to afford final phosphoramidites 31, 32 and 23.
  • Reaction Scheme 6, in Figure 9 shows the preparation of (2-deoxy- -D- ribofuranosyl)- 1 H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogues bearing guanidinoalkynyl substituates at the 3-position of the nucleobase.
  • the crude product was dissolved in DMF and pyridine, concentrated to remove moisture, then dissolved in anhydrous pyridine and added dimethylaminopyridine (100 mg, catalytic), 4,4'-dimethoxytrityl chloride (2.24 g, 6.6 mmol), and stirred for 72 hours. Concentrate and partition between ethyl acetate and water. Wash the aqueous layer with 10% citric acid, saturated NaHC0 3(aq) , brine, dry over MgSC1 ⁇ 4, and concentrate to an orange foam. Purify the crude product by flash chromatography using 33% acetone in dichloromethane to obtain 14 as a yellow-orange amorphous solid (3.5 g, 73% yield).
  • This example illustrates the preparation of 3-aminoalkynyl-substituted 1 H- pyrazolo[3,4-d]pyrimidin-4(5H)-one phosphoramidites 18, 20, 22 and 24 and their incorporation into oligonucleotides.
  • Phosphoramidites 31, 32 and 33 were prepared using the procedure described for compounds 18, 20, 22 and 24.
  • Oligonucletides were prepared in 200 nmol scale from commercially available 3 '-phosphoramidites and solid supports (Glen Research, Inc.) following standard synthesis and deprotection protocol for DNA synthesizer (Applied Biosystems, Model 3900). 5'-Dimethoxytritylated oligonucleotides were purified by RP-HPLC (C-18, 0.1 M triethylammonium bicarbonate/acetonitrile), detritylated and re-purified. Experimental ESI mass spectral data for all oligonucleotides corresponded to calculated values.
  • This example illustrates the preparation of oligonucleotides containing 3- guanidinooalkyn l-substi tuted (2-deoxy-P-D-ribofuranosyl)- lH-pyrazolo[3,4-d]pyrimidin- 4(5H)-ones I4g and I6g.
  • Oligonucleotides that contained modifications I4g and I6g were synthesized according to Roig, V.; Asseline, U. J. Am. Chem. Society, 2003, 125, 4616-4617 by a treatment of amine-modified oligonucleotide precursors (25 nmol) with 0. 1 1 M solution of 1 -pyrazole- l -carboxamidine hydrochloride in 1 M Na 2 C0 3 (30 ul) for 1 day at room temperature.
  • the modified oligonucleotides were purified by C 18 reverse phase chromatography in a gradient of acetonitryl in 0.1 M triethylammonium bicarbonate buffer. The identity and purity of all modified oligonucleotides were confirmed by mass spectroscopy.
  • This example illustrates the performance of 3-substituted-l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one bases of the invention compared to that of hypoxanthine when substituted in oligonucleotides in duplex formation.
  • the melting temperature was determined by combining the 3-alkynyl- 1 H- pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue-containing oligonucleotides with natural complements, with one experiment each for inosine analogue paired with adenine, cytosine, guanidine, and thymidine.
  • the oligonucleotides containing inosine analogue were measured in three formats: 1 ) inosine analogue in the 6 position, measured from the 5 * -end of the oligonucleotide; 2) inosine analogue in the 9 position, measured from the 5 '-end of the oligonucleotide; 3) inosine analogue in both the 6 and positions, measured from the ' -end of the oligonucleotide.
  • Oligonucleotides were combined in equimolar 2 ⁇ concentrations in buffer containing 100 niM NaCl. 10 niM MgCl 2 , and 10 m Na-PIPES (pH 7). The solutions in 1 cm cuvettes were brought to 80 °C briefly then the temperature lowered to 15 °C. Measurements were conducted on a Cary Bio 400 UV-Vis spectrophotometer equipped with a thermal peltier cell block and temperature probe. The temperature was ramped at a rate of 0.8 °C/min from 15 to 75 °C with the wavelength monitored at 268 nm. The melting temperature was calculated as the midpoint between the baselines of the associated and dissociated portions of the melting curve.
  • duplexes containing analogues of the invention substituted for a base opposite A, T and C in a generally more stable than the duplex the duplexes containing deoxyinosine.
  • G similar T m s are observed for both deoxyinosine and the analogues of the invention.
  • aminobutynyl-substituted analog (107) stabilizes A,T and C pairs greater than any other studied analogues.
  • SDS Applied Biosystems, Foster City, CA
  • 50 cycles of a two step PCR 95°C for 15 s, 65°C or 70° for 30 s profile was run. after an initial 1 5 min at 95 °C.
  • Commercially available 2x Qiagen QuantiTect Probe PCR Master mix (Qiagen cat.# 204345) was used. Final concentration of both primers was 0.5 ⁇ .
  • Each 20 ⁇ reaction contained 10 ng of template DNA. Routinely DNA samples were tested in triplicates using a 384-well plate,
  • An adenovirus assay was developed using a fluorogenic reverse flap primer (US Application No. 2007-0048758) which contained a minor groove binder ligand (DPI 3 ) and fluorescein (FAM) as a fluorescent label.
  • the forward primer contained deoxyinosine, hydroxybutynyl (104) or aminobutynyl (I07)-substituted l H-pyrazolo[3,4- d]pyrimidin-4(5H)-one nucleosides in various positions ( Figure 14 and 15).
  • An unmodified forward primer was also utilized as a positive control.
  • This example illustrates the ability of multiple substitutions of of the 3-(hydroxybutynyl)-lh-pyrazolo[3,4-d]pyrimidin-4(5h)-one and 3-(aminobutynyl)-l h-pyrazolo[3,4-d]pyrimidin-4(5h)-one -substituted PCR primers to efficiently participate in the amplification of Meticillin-resistant Staphylococcus aureus LGA251 target.
  • PCR is performed using the final concentrations of the assay components in the reaction mixture is the dT(8)-AP593 passive control. 0.035 ⁇ , forward primer 1 .260 ⁇ , reverse primer 0.500 ⁇ , probe 0.200 ⁇ I X enhancer, I X Tfi PCR Master Mix (Life Science Technologies, Inc) contains all the reagents necessary to perform PCR including uracil-N-glycosylase (UNG). Twenty microliters of the mixture was introduced in a 96 well PCR plate with 1 ⁇ , of sample nucleic acid. The plate was sealed with MicroAmp® Optical Adhesive Film (Applied Biosystems, Foster City, CA) and then centrifuged to collect the assay solution in the bottom of the plate well. The assay was then performed in an ABI 7500 DX Fast Block Real-time PCR machine with the protocol shown in Table 1 below.
  • UNG uracil-N-glycosylase
  • duplex containing the 3-(hydroxybutynyl)- l H- pyrazolo[3,4-D]pyrimidin-4(5H)-one is-substituted for three A's and two T's, amplified well at 56° and 60°C with cts of 36 and 36 respectively.
  • the same substitutes with inosine only amplified at 56°C with a Ct of 42.
  • PCR was performed as described in Example 9.
  • the target sequence and primer sequences are shown in Figure 17.
  • the amplicons obtained from amplification with the natural primer, the F(dl) primer and the F(I07) primer were submitted for sequencing analysis.
  • the sequences of the amplicons generated by these primers are shown in Figure 17.
  • the amplicon generated by the natural primer incorporated two As complementary to the Ts in the primers.
  • DNA polymerase incorporated a C complementary to the inosine and the aminobutylinosine.
  • pyrene modified nucleotides to increase duplex stability is understood in the art.
  • Kumar et al. attached pyrene to the 5 " -position of thymidine or to the 2' -posit ion of uridine with either a rigid tria/ole- or more flexible tria/ole methylene linkers.
  • the pyrene substituted to the 5 '-position tended to intercalate with increase stability while substitution with the more rigid linker tended to decrease duplex stability.
  • C2'-Pyrene- functionalized triazole-linked DNA/RNA universal hybridization probes were evaluated as promising universal probes (Sau & Hrdlica). Pyrene directly attached to the 1 ' -position of the deoxyribofuranose ring demonstrated selective and stable base pairing without hydrogen bonding when incorporated to an oligonucleotide (Matray & Kool).
  • the pyrene-substituted inosine phosphoramidite 35 was synthesized in two steps from intermediate 25 as shown in Figure 18. .
  • a general procedure for the synthesis of oligonucleotides containing pyrene and acridine moieties is shown in Figure 19.
  • a similar approach yielded the acridine derivative (107-Acr).
  • This example illustrates the preparation of 3-aminoalkynyl-substituted (2- deoxy-P-D-ribofuranosyl)-l H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 5 '-phosphoramidite 35 and their incorporation into oligonucleotides.
  • Oligonucletides were prepared in 200 nmol scale from commercially available 3 ' -phosphoramidites and solid supports (Glen Research, Inc.) following standard protocol for DNA synthesizer (Applied Biosystems, Model 3900). Oligonucleotides containing monomers 35 were cleaved from solid support and deprotected by ammonia hydroxide treatment (2h, +70oC). 5 ' - Dimethoxytrity lated oligonucleotides were purified by RP-HPLC (C- 18, 0.1 M triethylammonium bicarbonate/acetonitrile), detritylated and re- purified. Experimental ESI mass spectral data for all oligonucleotides corresponded to calculated values. EXAMPLE 13
  • This example illustrates the post-synthetic preparation of oligonucleotides containing 3-aminobutynyl-(2-deoxy- -D-ribofuranosyl)-l H-pyrazolo[3.4-d]pyrimidin- 4(5H)-ones substituted with pyrene and acridine moieties.
  • Pentafluorophenyl trifluoroacetate 1 .1 eq was added to a solution of 1 - pyreneacetic acid or 1 -pyrenebutyric acid and triethylamine (1.5 eq) in DCM (5 ml/mmol). The resultant mixture was kept at room temperature for 1 h and concentrated in vacuo. The residue from the reaction of 1-pyreneacetic acid was diluted with ether, the obtained solid collected by filtration, washed with ether and dried in vacuo to give PFP 1 -pyreneacetate (54% yield), which was used without further purification.
  • Oligonucleotides that contained Pyrene and Acridine moieties were synthesized by a treatment of amine-modified oligonucleotide precursors (75 nmol) with solution of corresponding PFP-ester ( 1 ⁇ ) and triethylamine ( 14.4 ⁇ ) in dry DMSO (70 ⁇ ) for 1 day at room temperature.
  • the modified oligonucleotides were purified by CI 8 reverse phase chromatography in a gradient of acetonitryl in 0.1 M triethylammonium bicarbonate buffer. The identity and purity of all modified oligonucleotides were confirmed by mass spectroscopy.
  • This example evaluates the effect on melting temperature of a number of oligonucleotides when substituted with inosine, 104, 107, 107-pyr and 107-acr, shown below:
  • inosine 104, 107, 107-pyr and 107-acr are numbered respectively 1 to 5.
  • substitution of two bases by inosine substantially drops the T m s over that of the match.( Figure 23).
  • the substitution of 107, 107-pyr and 107-acr performs well compared to inosine to increase T m s substantially for all substitutions tested except for GG substitution.
  • the T m s are comparable to that of the match.
  • the increases in T m s of the GT and TG substitutions are noteworthy.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des analogues de 3-alcynyl inosine et leurs utilisations en tant que bases universelles. Les analogues d'inosine peuvent être incorporés dans des amorces et des sondes d'acide nucléique. Lesdits analogues ne déstabilisent pas de façon significative des duplex d'acides nucléiques. Par conséquent, les nouvelles amorces et sondes d'acide nucléique incorporant les analogues d'inosine peuvent être utilisées dans divers procédés. Les analogues fonctionnent remarquablement bien, de façon inattendue, en tant que bases universelles. Lesdits analogues stabilisent non seulement des duplex sensiblement plus que l'hypoxanthine contre A, C, T et G, mais ils sont également reconnus dans des amorces par les polymérases, ce qui permet d'assurer une amplification efficace.
EP14714494.3A 2013-03-14 2014-03-07 Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation Withdrawn EP2971086A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13/827,456 US8969003B2 (en) 2011-03-23 2013-03-14 Functionalized 3-alkynyl pyrazolopyrimidine analogues as universal bases and methods of use
PCT/US2014/021799 WO2014159063A1 (fr) 2013-03-14 2014-03-07 Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation

Publications (1)

Publication Number Publication Date
EP2971086A1 true EP2971086A1 (fr) 2016-01-20

Family

ID=50397297

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14714494.3A Withdrawn EP2971086A1 (fr) 2013-03-14 2014-03-07 Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation

Country Status (2)

Country Link
EP (1) EP2971086A1 (fr)
WO (1) WO2014159063A1 (fr)

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4351760A (en) 1979-09-07 1982-09-28 Syva Company Novel alkyl substituted fluorescent compounds and polyamino acid conjugates
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US5202231A (en) 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
US6270961B1 (en) 1987-04-01 2001-08-07 Hyseq, Inc. Methods and apparatus for DNA sequencing and DNA identification
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
EP1231282A3 (fr) 1990-12-06 2005-05-18 Affymetrix, Inc. Méthodes et compositions pour l'identification de polymères
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5766855A (en) 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5419966A (en) 1991-06-10 1995-05-30 Microprobe Corporation Solid support for synthesis of 3'-tailed oligonucleotides
US5512677A (en) 1991-08-13 1996-04-30 National Science Council 3-substituted methyl-2,3-dihydroimidazo 1,2-c!quinazoline derivatives, the preparation and use thereof
US5556752A (en) 1994-10-24 1996-09-17 Affymetrix, Inc. Surface-bound, unimolecular, double-stranded DNA
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US5801155A (en) 1995-04-03 1998-09-01 Epoch Pharmaceuticals, Inc. Covalently linked oligonucleotide minor grove binder conjugates
US5736626A (en) 1996-01-29 1998-04-07 The Perkin-Elmer Corporation Solid support reagents for the direct synthesis of 3'-labeled polynucleotides
AU2229699A (en) 1998-01-16 1999-08-02 Comsat Corporation Arithmetic coding-based facsimile compression with error detection
US7045610B2 (en) 1998-04-03 2006-05-16 Epoch Biosciences, Inc. Modified oligonucleotides for mismatch discrimination
US6949367B1 (en) 1998-04-03 2005-09-27 Epoch Pharmaceuticals, Inc. Modified oligonucleotides for mismatch discrimination
US6127121A (en) 1998-04-03 2000-10-03 Epoch Pharmaceuticals, Inc. Oligonucleotides containing pyrazolo[3,4-D]pyrimidines for hybridization and mismatch discrimination
US6727356B1 (en) 1999-12-08 2004-04-27 Epoch Pharmaceuticals, Inc. Fluorescent quenching detection reagents and methods
US20010053404A1 (en) 2000-05-12 2001-12-20 Powrie William Duncan Formulation and process for producing a universal fruit base for use in preparing non-settling, creamy, smooth, thick fruit beverages
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7319022B1 (en) 2004-08-13 2008-01-15 Epoch Biosciences, Inc. Amplification methods
US20070048758A1 (en) 2005-06-09 2007-03-01 Epoch Biosciences, Inc. Improved primer-based amplification methods
WO2012129547A1 (fr) * 2011-03-23 2012-09-27 Elitech Holding B.V. Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisées comme bases universelles et procédés d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2014159063A1 *

Also Published As

Publication number Publication date
WO2014159063A1 (fr) 2014-10-02

Similar Documents

Publication Publication Date Title
EP2689031B1 (fr) Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisées comme bases universelles et procédés d'utilisation
EP1144429B1 (fr) Hybridation et discrimination du defaut de complementarite au moyen d'oligonucleotides conjugues a des molecules de liaison de petits sillons (mgb)
US8057997B2 (en) Nucleic acid binding compounds containing pyrazolo[3,4-D]pyrimidine analogues of purin-2,6-diamine and their uses
US7718374B2 (en) Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7566775B2 (en) N8- and C8-linked purine bases and structurally related heterocycles as universal nucleosides used for oligonucleotides hybridization
EP0628051B1 (fr) Utilisation de n-nucleosides fluorescents et de leurs analogues structurels fluorescents
JP4558932B2 (ja) ハイブリダイゼーション及び不一致識別のための、ピラゾロ［３，４−ｄ］ピリミジン含有オリゴヌクレオチド
JP4942484B2 (ja) 改良された感度および低バックグラウンドを備えるハイブリダイゼーションによるｄｎａ検出のための蛍光プローブ
US8969003B2 (en) Functionalized 3-alkynyl pyrazolopyrimidine analogues as universal bases and methods of use
WO2014186147A2 (fr) Pcr numérique à gouttelettes (ddpcr) utilisant de petites sondes courtes cannelées
US20090111100A1 (en) Minor groove binder - energy transfer oligonucleotides and methods for their use
EP2270015B1 (fr) Réactif d'hybridation sélectif de l'arn et son utilisation
WO2014159063A1 (fr) Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisés utilisés en tant que bases universelles et procédés d'utilisation
EP1138688A1 (fr) Bases puriniques liées en position N8- ou C8- comme nucléosides universelles utilisées pour hybridization avec oligonucléotides
WO2011034895A1 (fr) Compositions, procédés et utilisations impliquant des analogues de nucléotides
WO2001023611A2 (fr) Procedes

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20150916

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20160524