Functionalized 3-alkynyl pyrazolopyrimidine analogues as universal bases and methods of use

摘要

3-alkynyl inosine analogs and their uses as universal bases are provided. The inosine analogs can be incorporated into nucleic acid primers and probes. They do not significantly destabilize nucleic acid duplexes. As a result, the novel nucleic acid primers and probes incorporating the inosine analogs can be used in a variety of methods. The analogs function unexpectedly well as universal bases. Not only do they stabilize duplexes substantially more than hypoxanthine opposite A, C, T, and G but they are also recognized in primers by polymerases, allowing efficient amplification.

主权项

1. A method for the preparation of mismatched nucleic acid duplexes comprising:
a) providing a mixture including a sample containing one or more target nucleic acids and at least one oligonucleotide that is substantially complementary to the target nucleic acid, wherein said oligonucleotide comprises at least one 3-alkynyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue, and forms a duplex with target nucleic acid of substantially the same stability, regardless which natural nucleic acid base is positioned opposite to the 3-alkynyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one base; and b) incubating the mixture under hybridization conditions, wherein the 3-alkynyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one analogue is substituted with pyrene or acridine.