US 6,143,877 A · Oligonucleotides including pyrazolo[3,4-D]pyrimidine bases, bound in double stranded nucleic acids

Abstract

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.

Claims29

Dependent claims

A method of forming a triplex from a duplex nucleic acid having in one strand thereof a target sequence that consists essentially of nucleotides of purine bases and from an oligonucleotide that includes a substantially continuous sequence of at least 6 nucleotides, which sequence is complementary in the triplex forming sense pursuant to the G/T or A/G recognition motif to the target sequence in the duplex nucleic acid, at least one guanine base containing nucleotide in the substantially continuous sequence of the oligonucleotide being replaced by the nucleotide of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, the process comprising the step of:

contacting the oligonucleotide with the duplex nucleic acid.

A triplex comprising a triplex forming oligonucleotide that is not a homopolymer and that includes a substantially continuous sequence of at least 6 nucleotides, which sequence is complementary in the triplex forming sense pursuant to the G/T or A/G recognition motif to a target sequence in duplex nucleic acid, at least one guanine base containing nucleotide in the substantially continuous sequence of the triplex forming oligonucleotide being replaced by the nucleotide of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, the oligonucleotide having the formula ##STR11## wherein B is an independently selected heterocyclic base at least one of which is said 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one;

R.sub.1 independently is H, O-C.sub.1-6 alkyl, OC.sub.2-6 alkenyl, or F;
R.sub.2 independently is H or C.sub.1-6 alkyl;
r is an integer between approximately 5 to approximately 79;
X.sub.1 is independently O or S;
X.sub.2 is independently O or NH;
r' is an integer between 0 and 5;
r" is an integer between 0 and 1;
Z and Z' each independently represent an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the Z group and/or the Z' group respectively, to a heterocyclic base of the oligonucleotide;
W.sub.1 is H, a phosphate or thiophosphate group, a tail moiety, or W.sub.1 is selected from an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the W.sub.1 group to the 5'-end of the oligonucleotide;
W.sub.2 is H, a phosphate or thiophosphate group, a tail moiety, or W.sub.2 is selected from an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the W.sub.1 group to the 3'-end of the oligonucleotide, where at least one of said B groups represents a 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one-1-yl radical, and
said duplex nucleic acid including the target sequence.

8 more elements

A triplex comprising a triplex forming oligonucleotide that is not a homopolymer and that includes a substantially continuous sequence of at least 6 nucleotides, which sequence is complementary in the triplex forming sense pursuant to the G/T or A/G recognition motif to a target sequence in duplex nucleic acid, at least one guanine base containing nucleotide in the substantially continuous sequence of the triplex forming oligonucleotide being replaced by the nucleotide of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, the oligonucleotide having the formula ##STR12## wherein B is an independently selected heterocyclic base at least one of which is said 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one;

X.sub.2 independently is O or NH;
r is an integer between approximately 5 to approximately 79;
r' is an integer between 0 and 5;
r" is an integer between 0 and 1;
Z and Z' each independently represent an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the Z group and/or the Z' group respectively, to a heterocyclic base of the oligonucleotide;
W.sub.1 is H, a phosphate or thiophosphate group, a tail moiety, or W.sub.1 is selected from an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the W.sub.1 group to the 5'-end of the oligonucleotide;
W.sub.2 is H, a phosphate or thiophosphate group, a tail moiety, or W.sub.2 is selected from an intercalator group, a lipophilic group, a minor groove binder group, a reporter group, a chelating group, or a cross-linking group, each of said groups including a linker arm that connects the W.sub.1 group to the 3'-end of the oligonucleotide, where at least one of said B groups represents a 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one-1-yl radical, and
said duplex nucleic acid including the target sequence.

5 more elements

Dependencies are inferred from the claim text.

Oligonucleotides including pyrazolo[3,4-D]pyrimidine bases, bound in double stranded nucleic acids

Life of this patent

Oligonucleotides including pyrazolo[3,4-D]pyrimidine bases, bound in double stranded nucleic acids

Life of this patent

Abstract

Claims29