Formation of triple helix complexes for the detection of double stranded DNA sequences using oligomers which comprise an 8-modified purine base
AssigneeThe Johns Hopkins University
InventorsPaul S. Miller, Purshotam Bhan
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Abstract
A specific segment of double stranded DNA may be detected or recognized by formation of a triple helix structure using an Oligomer comprising an 8-modified purine base such as 8-oxo-adenine or 8-oxo-guanine to form a detectable triple helix complex. Function or expression of double stranded DNA segments may be prevented by triple helix formation. Novel Oligomers comprising an 8-modified purine base are useful in triple helix formation, and may be optionally derivatized with DNA modifying groups.
Claims30
A method of detecting a specific segment of double stranded nucleic acid without interrupting base-pairing of said double stranded nucleic acid which comprises contacting said nucleic acid segment with an Oligomer and detecting said segment, wherein said Oligomer
- (1) comprises at least one 8-oxo-adenine or 8-oxo guanine nucleosidyl unit, and
- (2) is sufficiently complementary to said nucleic acid segment, or a portion of the nucleotide sequences thereof, to form a triple helix structure wherein
- (a) one strand of the nucleic acid segment comprises a polyhomopurine sequence of at least 7 adjacent purine bases,
- (b) the Oligomer is parallel with respect to 5' to 3' orientation to the strand of the nucleic acid segment having the polyhomopurine sequence,
- (c) guanine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of
- (i) 8-oxo-adenine, and
- (ii) cytosine or a cytosine analog, either of which is protonated at N-3 of cytosine under physiological pH so as to hydrogen bond with guanine, and
- (d) adenine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of 8-oxo-guanine, thymine and uracil.
A triple helix structure which comprises a double stranded nucleic acid sequence and an Oligomer which
- (1) comprises at least one 8-oxo-adenine or 8-oxo guanine nucleosidyl unit, and
- (2) is sufficiently complementary to read a polyhomopurine sequence in one strand of the nucleic acid sequence and to form triplets, wherein
- (a) the Oligomer is parallel with respect to 5' to 3' orientation to the strand having the polyhomopurine sequence,
- (b) guanine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of
- (i) 8-oxo-adenine, and
- (ii) cytosine or a cytosine analog, either of which is protonated at N-3 of cytosine under physiological pH so as to hydrogen bond with guanine, and
- (c) adenine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of 8-oxo-guanine, thymine and uracil.
An Oligomer capable of reading a polyhomopurine sequence of one strand of a double stranded nucleic acid sequence,
- (1) wherein said Oligomer has at least one 8-oxo-adenine nucleosidyl unit,
- (2) guanine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of
- (a) 8-oxo-adenine, and
- (b) cytosine or a cytosine analog, either of which is protonated at N-3 of Cytosine under physiological pH so as to hydrogen bond with guanine, and
- (3) adenine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of 8-oxo-guanine, thymine and uracil.
A method of making a synthetic Oligomer which binds to a specific segment of double stranded nucleic acid to form a triple helix structure by hydrogen bonding thereto, said method comprising the steps of:
- (a) identifying a double stranded nucleic acid target segment wherein the nucleoside sequence of one strand of the nucleic acid segment comprises a polyhomopurine sequence of at least 7 adjacent purine bases;
- and
- (b) synthesizing said synthetic Oligomer complementary to said target sequence, said synthetic Oligomer including at least one 8-oxo-adenine or 8-oxo-guanine nucleosidyl unit,
- wherein said synthetic Oligomer has a nucleosidyl unit having a base selected from the group consisting of
- (i) 8-oxo-adenine, and
- (ii) cytosine or a cytosine analog, either of which is protonated at N-3 of cytosine under physiological pH when the complementary location in the polyhomopurine sequence has a G, and
- wherein such synthetic Oligomer has a nucleosidyl unit having a base selected from the group consisting of 8-oxo-guanine, thymine and uracil when the complementary location in the polyhomopurine sequence has an A, and
- wherein said Oligomer is orientated 5' to 3' and binds parallel to the strand having the polyhomopurine sequence.
An Oligomer capable of reading a polyhomopurine sequence of one strand of a double stranded nucleic acid sequence,
- (1) wherein said Oligomer has at least one 8-oxo-adenine or 8-oxo-guanine nucleosidyl unit,
- (2) guanine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of
- (a) 8-oxo-adenine, and
- (b) cytosine or a cytosine analog, either of which is protonated at N-3 of cytosine under physiological pH so as to hydrogen bond with guanine,
- (3) adenine in the polyhomopurine sequence is read by a base in the Oligomer selected from the group consisting of 8-oxo-guanine, thymine and uracil, and
- (4) said Oligomer comprises an alkyl- or aryl-phosphonate Oligomer.
Dependencies are inferred from the claim text.