Epoch Pharmaceuticals, Inc.

Microprobe, Inc.

Epoch Biosciences, Inc.

Elitech Holding B.v.

Other

Yale University

Rich B Meyer, Jr

Igor V Kutyavin

Eugeny Alexander Lukhtanov

Howard B Gamper

Alexander A Gall

Irina Aleksandrovna Afonina

Joel Hedgpeth

Evgeniy S Belousov

Michael W Reed

Charles R Petrie

John C Tabone

Nicolaas M Vermeulen

B Melina Cimler

Gerald D Hurst

Yung-Chi Cheng

Jeffrey Van Ness

James H Zhou

Balakrishna S Pai

Arthur D Broom

A David Adams

Howard B Gamper, Jr

J Ansel Wald

Jeffrey V Ness

Jinsuk Woo

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPIgroup resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo&lsqb;3,2-e&rsqb;indole-7-carboxylate (CDPI ), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Minor groove binding molecules are covalently bound to oligonucleotides which in their base sequence are complementary to a target sequence of single stranded or double stranded DNA, RNA or hybrids thereof. The covalently bound oligonucleotide minor groove binder conjugates strogly bind to the target sequence of the complementary strand.

Covalently linked oligonucleotide minor groove binder conjugates

Oligonucleotides in which one or more purine residues are substituted by pyrazolo&lsqb;3,4-d&rsqb;pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo&lsqb;3,4-d&rsqb;pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo&lsqb;3,4-d&rsqb;pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.

Oligonucleotides containing pyrazolo&lsqb;3,4-d&rsqb;pyrimidines for hybridization and mismatch discrimination

Chemically modified oligonucleotides (ODNS) are complementary, either in the sense of the classic &ldquo;four letter code&rdquo; recognition motif, or in the sense required for triple strand formation based on the more limited &ldquo;two letter code recognition motif&rdquo;, to a target sequence of double stranded DNA of an invading cell, organism or pathogen, such as a virus, fungus, parasite, bacterium, malignant cell, or any duplex DNA which is desired to be broken into segments for the purpose of &ldquo;mapping&rdquo;. The ODNs have cross-linking agents covalently attached at least to two different sites of the ODN. Alternatively, the cross-linking agent which is attached to one site on the ODN has two cross-linking functionalities, and therefore in effect comprises two cross-linking agents. The cross-linking agent typically includes a linker arm (such as an alkyl, alkoxy, aminoalkyl or amidoalkyl chain) and a reactive group which, after triple strand formation with the target sequence of DNA, is capable of reacting with the target DNA to form a covalent bond therewith. Each cross-linking agent of the novel modified ODNs is capable of forming a covalent bond with the target DNA. As a result of the covalent bond formation between the modified ODN and both strands of the target DNA sequence, replication and expression of the target DNA sequence is inhibited. Alternatively the duplex DNA is selectively cleaved with enzymes or amino acids, at the alkylation sites for &ldquo;mapping&rdquo; or other investigative purposes.

Bifunctional Crosslinking oligonucleotides adapted for linking to a target sequence of duplex DNA

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo&lsqb;3,2-e&rsqb;indole-7-carboxylate (CDPI,), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI,group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

Methods and compositions for efficient targeting and modification of target sequences in duplex DNA are provided, utilizing oligonucleotides or oligonucleotide compositions containing two domains. The first domain comprises an entity capable of recognizing a double-stranded DNA sequence. This can be a protein, peptide, antibiotic, minor groove binding agent or a nucleotide sequence capable of triplex formation The second domain, which is covalently joined to the first, is capable of recognizing a single-stranded DNA sequence. This second domain will most often be complementary, in the Watson-Crick sense, to a target sequence in the double-stranded nucleic acid. The second domain can optionally carry one or more modifying groups, capable of causing a mutation, a pre-mutagenic lesion, or some other type of heritable change in the target sequence.

Methods and compositions to facilitate D-loop formation by oligonucleotides

A triplex forming oligonucleotide is complementary pursuant to the G/T or A/G recognition motif to a homopurine, or substantially homopurine target sequence in double stranded nucleic acids, and at least one and preferably all of the guanine bases are replaced by their pyrazolo[3,4-d]pyrimidine analog, namely by 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one. The oliginucleotides containing the pyrazolo[3,4-d]pyrimidine analog of guanine exhibit a lesser degree of self-association, and lack the nucleophilic nitrogen atom in the 7 position of guanine. The latter feature results in a diminished extent of self-crosslinking in ODNs which also have a covalently attached cross-linking agent.

Oligonucleotides including pyrazolo[3,4-D]pyrimidine bases, bound in

A method for introducing a site-specific mutation into a target polynucleotide sequence is presented. The method involves the use of an oligonucleotide capable of binding to the target sequence, either by triplex formation (mediated by Hoogsteen, reverse Hoogsteen or equivalent base pairing) or by Watson/Crick base pairing (in the presence of a recombinase enzyme). The oligonucleotide of the invention is modified by the covalent attachment of one or more electrophilic groups. When a modified oligonucleotide is bound to its target sequence, the electrophilic group is able to interact with a nearby nucleotide in the target sequence, causing a modification to the nucleotide that results in a change in nucleotide sequence. Compositions used in the practice of the method are also disclosed.

Targeted mutagenesis in living cells using modified oligonucleotides

Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.

Oligonucleotides containing pyrazolo[3,4-D]pyrimidines for hybridization

Minor groove binding molecules are covalently bound to oligonucleotides which in their base sequence are complementary to a target sequence of single stranded or double stranded DNA, RNA or hybrids thereof. The covalently bound oligonucleotide minor groove binder conjugates strongly bind to the target sequence of the complementary strand.

Growing community of inventors

Bothell, WA, United States of America

Rich B Meyer, Jr