Oligonucleotides comprising a molecular switch
First Claim
1. A tripartite oligonucleotide consisting essentially of the following three components:
- (a) a nucleic acid anchor region complementary to a first sequence of nucleic acid residues of a target nucleic acid; and
(b) a bridging domain; and
(c) a binding domain,wherein said bridging domain and said binding domain form a switch domain,wherein said binding domain comprises 2-20 nucleic acid bases or analogs thereof complementary to said target nucleic acid and said binding domain has less affinity for said target nucleic acid than said anchor region,wherein said bridging domain is located between said anchor region and said binding domain and comprises 2-11 universal, generic or mismatched natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases that do not form a Watson-Crick hybridization complex with said target nucleic acid, wherein two or more universal or non-hydrogen bonding natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases in said bridging domain are juxtaposed, and wherein said universal or non-hydrogen bonding natural bases in said bridging domain substitute for bases complementary to nucleotide bases of said target nucleic acid and enhance sensitivity to the presence of a mismatch between the binding region of the switch domain and the target sequence,wherein said switch domain is able to discriminate between (i) a sequence of nucleic acid residues of said target nucleic acid that is complementary to said binding domain and (ii) a second mismatch sequence of nucleic acid residues of said target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;
under conditions wherein said anchor region (a) forms a stable duplex with said first sequence of nucleic acid residues of said target nucleic acid; and
wherein said target nucleic acid comprises one or more sequences selected from the group consisting of a sequence that is associated with a disease or condition, a sequence that is associated with an infectious organism, and a sequence comprising a genetic variation.
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Abstract
This invention relates to oligonucleotides comprising a molecular switch which may exist in an “open” or “closed” position. The molecular switch portion of the probe is particularly sensitive to the identity of sequences complementary to the molecular switch. Oligonucleotides containing a molecular switch are applicable to all kinds of hybridization processes. Due to the sensitivity of the switch domain of the oligonucleotide, probes containing a molecular switch are particularly useful in the identification of single point mismatches. More specifically, a portion, but not all, of the oligonucleotide becomes unbound from a mismatched target. The invention further relates to methods of using said oligonucleotides for research reagents, and clinical diagnostics. An exemplary oligonucleotide comprises a first hybridizable domain, a second bridging block domain, and a third binding domain.
33 Citations
36 Claims
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1. A tripartite oligonucleotide consisting essentially of the following three components:
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(a) a nucleic acid anchor region complementary to a first sequence of nucleic acid residues of a target nucleic acid; and (b) a bridging domain; and (c) a binding domain, wherein said bridging domain and said binding domain form a switch domain, wherein said binding domain comprises 2-20 nucleic acid bases or analogs thereof complementary to said target nucleic acid and said binding domain has less affinity for said target nucleic acid than said anchor region, wherein said bridging domain is located between said anchor region and said binding domain and comprises 2-11 universal, generic or mismatched natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases that do not form a Watson-Crick hybridization complex with said target nucleic acid, wherein two or more universal or non-hydrogen bonding natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases in said bridging domain are juxtaposed, and wherein said universal or non-hydrogen bonding natural bases in said bridging domain substitute for bases complementary to nucleotide bases of said target nucleic acid and enhance sensitivity to the presence of a mismatch between the binding region of the switch domain and the target sequence, wherein said switch domain is able to discriminate between (i) a sequence of nucleic acid residues of said target nucleic acid that is complementary to said binding domain and (ii) a second mismatch sequence of nucleic acid residues of said target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;
under conditions wherein said anchor region (a) forms a stable duplex with said first sequence of nucleic acid residues of said target nucleic acid; andwherein said target nucleic acid comprises one or more sequences selected from the group consisting of a sequence that is associated with a disease or condition, a sequence that is associated with an infectious organism, and a sequence comprising a genetic variation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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9. A tripartite oligonucleotide consisting essentially of the following three components:
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(a) a nucleic acid anchor region complementary to a first sequence of nucleic acid residues of a double-stranded target nucleic acid, and (b) a bridging domain; and (c) a binding domain, wherein said bridging domain and said binding domain form a switch domain, wherein said binding domain comprises 2-20 nucleic acid bases or analogs thereof fully complementary to a second sequence of said double stranded target nucleic acid, and wherein said binding domain has less affinity for the target sequence than said anchor region, wherein said bridging domain is located between said anchor region and said binding domain and comprises 2-11 universal, generic or mismatched natural bases or analogs thereof or a mixture of universal bases and non-hydrogen bonding natural bases that do not form a Watson-Crick hybridization complex with said target nucleic acid, wherein two or more universal or non-hydrogen bonding bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases in said bridging domain are juxtaposed, and wherein said universal or non-hydrogen bonding natural bases in said bridging domain substitute for bases complementary to nucleotide bases of said target nucleic acid and enhance sensitivity to the presence of a mismatch between the binding region of the switch domain and the target sequence; wherein said switch domain is able to discriminate between (i) a sequence of nucleic acid residues of said double-stranded target nucleic acid that is complementary to said binding domain and (ii) a second mismatch sequence of nucleic acid residues of said double-stranded target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;
under conditions wherein said anchor region (a) forms a stable triple-stranded nucleic acid with said first sequence of nucleic acid residues of said double-stranded target nucleic acid; andwherein said target nucleic acid comprises one or more sequences selected from the group consisting of a sequence that is associated with a disease or condition, a sequence that is associated with an infectious organism, and a sequence comprising a genetic variation. - View Dependent Claims (36)
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18. An oligonucleotide comprising:
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(a) a nucleic acid anchor region complementary to a first sequence of nucleic acid residues of a target nucleic acid; and (b) a switch domain comprising at least one bridging domain and at least one binding domain, wherein said binding domain comprises 2-20 nucleic acid bases or analogs thereof complementary to said target nucleic acid and said binding domain has less affinity for said target nucleic acid than said anchor region, wherein said bridging domain is located between said anchor region and said binding domain and comprises 2-11 universal, generic or mismatched natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases that do not form a Watson-Crick hybridization complex with said target nucleic acid, wherein two or more universal or non-hydrogen bonding natural bases or analogs thereof or a mixture of universal and non-hydrogen bonding natural bases in said bridging domain are juxtaposed, and wherein said universal or non-hydrogen bonding natural bases in said bridging domain substitute for bases complementary to nucleotide bases of said target nucleic acid and enhance sensitivity to the presence of a mismatch between the binding region of the switch domain and the target sequence, wherein said switch domain is able to discriminate between (i) a sequence of nucleic acid residues of said target nucleic acid that is complementary to said binding domain and (ii) a second mismatch sequence of nucleic acid residues of said target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;
under conditions wherein said anchor region (a) forms a stable duplex with said first sequence of nucleic acid residues of said target nucleic acid; andwherein said tar et nucleic acid comprises one or more sequences selected from the group consisting of a sequence that is associated with a disease or condition, a sequence that is associated with an infectious organism, and a sequence comprising a genetic variation, wherein said oligonucleotide is attached to an electron conducting solid surface, and wherein the amount of hybridization to said target nucleic acid controls the amount of current flow.
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Specification