Non-aggregating non-quenching oligomers comprising nucleotide analogs; methods of synthesis and use thereof
AssigneeEpoch Biosciences, Inc.
InventorsAlexander Gall, Igor Kutyavin, Nicolaas Vermeulen, Robert Dempcy
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Abstract
The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.
Claims54
A composition comprising:
- wherein
- R1 and R2 are independently —H, —OH, —OR6, —SH, —NH2 or —NHR7;
- R3 is —H—CN, halogen (F, Cl, Br or I), or —R12—Y, wherein R12 is C1-C12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH2 or —SH;
- R6 is —H, —C6H5 (benzyl) or a diphenylcarbamoyl (DPC) group;
- R7 is a protecting group selected from the group consisting of 2-N-dimethylvinyl, benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyl, acetyl, and anisoyl (An) groups;
- and
- X is ═CH— or N—.
A composition comprising
- wherein
- R1 and R2 are independently —H, —OH, —OR6, —SH, —NH2 or —NHR7
- R3 is —H, —CN, halogen (F, Cl, Br or I), or —R12—Y, wherein R12 is C1-12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH2 or —SH;
- X is ═CH— or ═N—;
- R4 is —H or a protecting, group selected from the group consisting of tert-butyloxycarbonyl (tBoc), 4-methoxyphenyldiphenylmethyl (MMTr), isobutyryl (iBu) and 9-fluoronylmethyloxycarbonyl (Fmoc);
- R5 is —H or —C6F4H (TFP);
- R6 is —H, —C6H5 (benzyl) or a diphenylcarbamoyl (DPC) group;
- and
- R7 is a protecting group selected from the group consisting of 2-N-dimethylvinyl, benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyi, acetyl, and anisoyl (An) groups.
14-26. (canceled)
A method for detecting a target sequence in a polynucleotide wherein the improvement comprises:
- use of a probe, complementary to the target sequence, wherein one or more residues in the probe comprises abase analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines,
- such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and reduce the tendency of the probe to self-associate.
A method for the detection of a target sequence in a polynucleotide using a probe comprising a polymeric portion and a fluorescent portion, wherein the polymeric portion comprises a plurality of monomers;
- the improvement comprising
- one or more of the monomers in the probe comprises a base analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines;
- such that the base analogues retain the base-pairing specificity of the bases f which they. are substituted and reduce the tendency of the polymeric portion of the probe to quench emission from the fluorescent portion.
56-71. (canceled)
A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
- the use of the composition of claim 56 as a probe.
A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
- the use of the composition of claim 58 as a probe.
A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
- the use of the composition of claim 59 as a probe.
An oligomer microarray, comprising one or more oligomers according to claim 14.
A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:
- use of the oligomer of claim 14 as a probe.
A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:
- use of the composition of claim 56 as a probe.
A method for detecting a target sequence. in a polynucleotide by hybridization, wherein the improvement comprises:
- use of the composition of claim 57 as a probe.
Dependencies are inferred from the claim text.