A strongly pairing fifth base: oligonucleotides with a C-nucleoside replacing thymidine

Abstract

There are five canonical bases in DNA and RNA. Each base has its particular molecular recognition properties and base pairing strength. Thymine and uracil form only two hydrogen bonds when pairing with adenine, and duplexes rich in A:T base pairs are more labile than duplexes rich in C and G, making some sequences difficult to detect via hybridization in a genomic context. Here we report the synthesis of an ethynylmethylpyridone C-nucleoside, abbreviated ‘W’, that presents a similar recognition surface as thymidine in the major groove but pairs with A about as strongly as C pairs with G. A phosphoramidite building block was synthesized that allows for incorporation of W residues via automated synthesis in high yield. Melting point increases over duplexes containing T:A pairs of up to 17.5°C, or up to 5.8°C per residue were measured for oligonucleotides containing W. Further, the new base shows excellent fidelity, with a single mismatched G opposite W causing a melting point depression of up to 20.5°C. The strongly pairing replacement for thymidine is only slightly larger than its natural counterpart and performs well in different sequence contexts. It can be used to target weakly pairing A-rich sequences in biological studies.