Oligonucleotide N3'.fwdarw.P5' phosphoramidates: hybridization and nuclease resistance properties
AssigneeLynx Therapeutics, Inc.
InventorsSergei M. Gryaznov, Ronald G. Schultz, Jer-Kang Chen
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Abstract
Modified oligonucleotides 3'-NHP(O)(O.sup.-)O-5' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.
Claims40
A method of hybridizing an oligodeoxyribonucleotide to an RNA target, comprising:
- (a) forming an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages, wherein:
- (i) at least 50% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'.fwdarw.P5' phosphoramidate inter-subunit linkages of the formula:
- 3'-NH-P(.dbd.O)(-O.sup.-)-O-5';
- and
- (ii) said oligodeoxyribonucleotide has at least 11 nucleotides;
- and
- (b) contacting the oligodeoxyribonucleotide with said RNA target to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said RNA target.
A kit for the isolation of a target RNA from a sample, the kit comprising:
- an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages wherein at least 50% of the inter-subunit linkages of said oligodeoxyribonueleotide are N3'.fwdarw.P5' phosphoramidate inter-subunit linkages of the formula:
- 3'-NH-P(.dbd.O)(-O.sup.-)-O-5'
- wherein
- said oligodeoxyribonucleotide hybridizes to said target RNA, and
- said oligodeoxyribonucleotide has at least 11 nucleosides.
A diagnostic method to detect in a sample the presence of an RNA having a selected target sequence comprising:
- (a) contacting an oligodeoxyribonucleotide with said RNA to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said selected target sequence, the oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits and inter-subunit linkages wherein:
- (i) at least 50% of the inter-subunit linkages or said oligodeoxyribonucleotide are N3'.fwdarw.P5' phosphoramidate inter-subunit linkages of the formula:
- 3'-NH-P(.dbd.O)(-O.sup.-)-O-5',
- and
- (ii) said oligodeoxyribonucleotide has at least 11 nucleosides:
- and
- (b) detecting the presence of said hybridization complex, thereby detecting the presence of said RNA.
A method of hybridizing an oligodeoxyribonucleotide to an DNA target, comprising:
- (a) forming an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages, wherein:
- (i) 100% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'.fwdarw.P5' phosphoramidate inter-subunit linkages of the formula:
- 3'-NH-P(.dbd.O)(-O.sup.-)-O-5';
- and
- (ii) said oligodeoxyribonucleotide has at least 11 nucleotides;
- and
- (b) contacting the oligodeoxyribonucleotide with said DNA target to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said DNA target.
A diagnostic method to detect the presence of a single-stranded DNA having a selected target sequence in a sample, comprising:
- (a) contacting an oligodeoxyribonucleotide with said single-stranded DNA to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said selected target sequence, the oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits and inter-subunit linkages wherein:
- (i) 100% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'.fwdarw.P5' phosphoramidate inter-subunit linkages of the formula:
- 3'-NH-P(.dbd.O)(-O.sup.-)-O-5',
- and
- (ii) said oligodeoxyribonucleotide has at least 11 nucleosides;
- and
- (b) detecting the presence of said hybridization complex, thereby detecting the presence of said DNA.
A method of hybridizing an oligodeoxyribonucleotide to a polynucleotide target sequence, the method comprising:
- forming an oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits joined by inter-subunit linkages defined by the formula:
- ##STR1## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48;
- and
- (b) contacting the oligodeoxyribonucleotide with said polynucleotide target sequence to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said polynucleotide target sequence.
A kit for the isolation of a target polynucleotide from a sample, comprising:
- an oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits joined by inter-subunit linkages and defined by the formula:
- ##STR2## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48 and wherein said oligodeoxyribonucleotide hybridizes to said target polynucleotide.
A diagnostic method to detect the presence of a polynucleotide having a selected target sequence in a sample comprising:
- (a) contacting an oligodeoxyribonucleotide with said polynucleotide to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said target sequence, wherein said oligodeoxyribonucleotide comprises a defined sequence of nucleoside subunits joined by inter-subunit linkages defined by the formula:
- ##STR3## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48;
- and
- (b) detecting the presence of said hybridization complex, thereby detecting said polynucleotide.
Dependencies are inferred from the claim text.