Electroanalysis of unnatural base pair content in plasmid DNA generated in a semi-synthetic organism

Abstract

While standard Watson-Crick base pairing normally involves hydrogen bonds that join size complementary nitrogenous heterocycles, certain unnatural base pairs (UBP) can support replication, transcription, and translation, including these in semi-synthetic organisms (SSO), using size complementarity alone. In this paper we show that the UBPs can be analyzed electrochemically as components of ribo- or 2′-deoxyribonucleosides, corresponding (2’-deoxy)triphosphates, as well as incorporated into DNA. Here, performance of the electrochemical methods profits from the ability of the unnatural bases 5SICS and TPT3 adsorbed at the surface of a mercury electrode to catalyze hydrogen evolution in acidic media. This allows semi-quantitative detection of single UBP within plasmid DNA generated in a SSO. This electrochemical approach provides new way for direct, fast, and low cost UBP analysis and can supplement or replace currently used methods of UBP detection.