Deoxyuridine triphosphates modified with tyrosine or tryptophan aromatic groups for direct electrochemical detection of double-stranded DNA

Abstract

Deoxyuridine triphosphates (dUTP) modified with tyrosine or tryptophan aromatic groups attached through –CH=CH–CH2–NH–C(O)–(CH2)n– linker at the C5 position of the pyrimidine ring and the corresponding products of polymerase chain reaction (PCR) were studied by cyclic and square wave voltammetry on carbon screen printed electrodes. A strong effect of the additional functional groups on the electrochemical activity of nucleotides was revealed. In particular, 5-aminoallyl-2′-deoxyuridine-5′-triphosphates modified with indole acetic, indole-3-propionic, indole-4-butyric, or 4-hydroxyphenylacetic acids demonstrated novel well-defined oxidation peaks at 0.5–0.7 V, similar to tryptophan or tyrosine amino acids, respectively. The oxidation potential maxima for dUTP derivatives under study were about 0.2–0.3 V less positive than the oxidation potential of dGTP (the most easily oxidizable nucleotide). Moreover, dUTP derivatives modified with tyrosine or tryptophan aromatic groups were incorporated by PCR into dsDNA fragments (amplicons) instead of dTTP. The PCR-generated dsDNA fragments with modified nucleotides were detected through the oxidation of their eleсtroactive ‘labels’ at micromolar concentrations, while no oxidation peaks were observed for unmodified amplicons under the same conditions. The tyrosine and tryptophan aromatic groups well complement the existing palette of electroactive tags for direct detection of nucleic acids. Compared to other electroactive ‘labels’, the main advantage of the developed oxidizable moieties is a good compatibility with polymerase enzymes including those used in PCR. In addition, the demonstrated signal recording procedure for modified amplicons on disposable carbon electrodes renders itself suitable for in situ analysis of biological samples for biochemical and medical applications.