Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11](1-)), [3,3'-iron-bis(1,2-dicarbollide)](-) (FESAN, [Fe(C2B9H11)(2)](2-)) or [3,3'-cobalt-bis(1,2-dicarbollide)](-) (COSAN, [Co(C2B9H11)(2)](2)(-)) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dN(X)TPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Automated summary

A series of modified nucleoside triphosphates bearing dicarba-nido-undecaborate or metal bis(dicarbollide) groups were prepared and utilized for DNA synthesis and redox labeling. The combination of these redox labels allowed for the first fully orthogonal redox coding of all four canonical nucleobases, enabling determination of nucleobase composition of short DNA stretches with electrochemical readout in one simple primer extension experiment.