Use of Oligonucleotides with Modified Bases in Hybridization of Nucleic Acids
First Claim
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1. A method of inhibiting expression of a target nucleic acid comprising:
- contacting a target nucleic acid of known sequence with a modified oligonucleotide having a sequence of nucleobases that is at least partly complementary to a strand of said target nucleic acid, under conditions that permit hybridizing of the modified oligonucleotide to a strand of thc target nucleic acid,wherein the hybridized modified oligonucleotide inhibits expression of the target nucleic acid,wherein the modified oligonucleotide comprises from 5 to 150 nucleobases, andwherein at least one of the nucleobases of the modified oligonucleotide is a modified nucleobase selected from the group consisting of;
5-mercaptocytosine, 5-mercaptouracil, 8-mercaptoguanine, 8-mercaptoadenine, 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine and 8-hydroxyguanine.
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Abstract
The invention is concerned with the use of oligonucleotide analogs that contain specifically modified DNA bases to be used in hybridization of nucleic acids, polymerase chain reaction (PCR) and siRNA-mediated gene silencing (RNAi).
148 Citations
35 Claims
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1. A method of inhibiting expression of a target nucleic acid comprising:
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contacting a target nucleic acid of known sequence with a modified oligonucleotide having a sequence of nucleobases that is at least partly complementary to a strand of said target nucleic acid, under conditions that permit hybridizing of the modified oligonucleotide to a strand of thc target nucleic acid, wherein the hybridized modified oligonucleotide inhibits expression of the target nucleic acid, wherein the modified oligonucleotide comprises from 5 to 150 nucleobases, and wherein at least one of the nucleobases of the modified oligonucleotide is a modified nucleobase selected from the group consisting of;
5-mercaptocytosine, 5-mercaptouracil, 8-mercaptoguanine, 8-mercaptoadenine, 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine and 8-hydroxyguanine. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 26, 27, 28, 29, 30)
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11. A method of detecting a target nucleic acid with a modified oligonucleotide comprising:
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contacting a target nucleic acid with a modified oligonucleotide under conditions that permit hybridizing of the modified oligonucleotide to a strand of said target nucleic acid, wherein the modified oligonucleotide comprises a sequence of nucleobases that is at least partly complementary to a sequence of the strand of the target nucleic acid, and wherein at least one of the nucleobases of the modified oligonucleotide is a modified nucleobase selected from the group consisting of;
5-mercaptocytosine, 5-mercaptouracil, 8-mercaptoguanine, 8-mercaptoadenine, 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine and 8-hydroxyguanine; anddetecting the target nucleic acid by detecting the modified oligonucleotide hybridized to the strand of the target nucleic acid. - View Dependent Claims (12, 13, 14, 15, 24, 34)
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16. A method of polymerase chain reaction (PCR) comprising:
contacting a template nucleic acid with a modified oligonucleotide comprising a sequence sufficiently complementary to a portion of the template nucleic acid to allow hybridization of the modified oligonucleotide with the template nucleic acid under PCR annealing conditions, wherein the hybridized modified oligonucleotide serves as a PCR primer under PCR amplification conditions to generate a first strand PCR product, and wherein the modified oligonucleotide comprises from 5 to 150 nucleobases, wherein at least one of the nucleobases is a modified nucleobase selected from the group consisting of;
5-mercaptocytosine, 5-mercaptouracil, 8-mercaptoguanine, 8-mercaptoadenine, 5-hydroxycylosine, 5-hydroxyuracil, 8-hydroxyadenine and 8-hydroxyguanine and;performing a polymerasc chain reaction (PCR) with the modified oligonucleotide and the template nucleic acid. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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31. In a method of amplification of a target nucleic acid, the method comprising annealing an oligonucleotide primer that is complementary to at least a portion of the target nucleic acid to the target nucleic acid, the improvement comprising using a modified oligonucleotide as the oligonucleotide primer, wherein the modified oligonucleotide comprises one or more modified nucleobases selected from the group consisting of:
- 5-mercaptocytosine, 5-mercaptouracil, 8-mercaptoguanine, 8-mercaptoadenine, 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine and 8-hydroxyguanine.
- View Dependent Claims (32, 33)
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35. An isolated nucleic acid consisting of a polynucleotide sequence selected from the group consisting of SEQ ID NO:
- 10, SEQ ID NO;
11, SEQ ID NO;
12, and SEQ ID NO;
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- 10, SEQ ID NO;
Specification