Deoxyuridine triphosphates modified with tyrosine aromatic groups for direct electrochemical detection of double-stranded DNA products of isothermal recombinase polymerase amplification

Abstract

A set of novel 2′-deoxyuridine-5′-triphosphate (dUTP) derivatives modified with tyrosine (Tyr) or tryptophan (Trp) aromatic groups (m-dUTP) was tested as bearers of electroactive ‘labels’ for double-stranded DNA (dsDNA) detection as well as proper substrates for use in recombinase polymerase amplification (RPA). The ‘labeled’ dUTP have demonstrated oxidation peaks at 0.50–0.65 V, similar to free Tyr or Trp. Among five m-dUTP tested in this study, merely two nucleotides modified with Tyr aromatic groups revealed good compatibility with RPA. The modified dsDNA amplicons, generated by RPA, have been detected at micromolar concentrations via the oxidation of electroactive ‘labels’, while no signals were registered under the same conditions for the control unmodified dsDNA. Therefore, the developed dUTP derivatives with Tyr aromatic groups appear as promising recombinase substrates for a further elaboration of on-site electrochemical assays for medical, ecological, or agricultural applications.

Keywords

• Nucleic acid
• Modified nucleotide
• Oxidation
• Tyrosine
• Isothermal amplification