Aminophenyl‐ and Nitrophenyl‐Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection

Abstract

Primer extension is used to incorporate labeled nucleoside triphosphates into oligonucleotides (ONs). Aminophenyl and nitrophenyl modifications serve as electrochemical labels that are detectable by either oxidation or reduction, respectively (see representation of the ON double strand and redox curves). The redox potentials of the labels differ depending on the nucleobase and respond to incorporation into ONs.

Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA

“…10). EBF and PhNO 2 yielded, in agreement with previous literature, 5,9 specific reduction signals around −0.95 V and −0.55 V, respectively. When the measurements were performed with ONs freshly adsorbed at the electrode and the initial potential (E i ) was set at 0.0 V, no interfering anodic signals of ONs modified with BF or PhNO 2 were detected in the anodic scan.…”

“…The directly linked A PT is more useful than the ethynyl-linked A EPT base where the second oxidation peak overlaps with oxidation of guanine. The PT-redox label is orthogonal to previously reported benzofurazane 9 and nitrophenyl labels 5 and can be used for multipotential redox coding of DNA bases. 12 The characteristic reversibility of the first PT signal and the irreversibility of the two-step oxidation process can be exploited for unambiguous identification of the PT label among other oxidizable moieties.…”

“…The syntheses started from commercial 2′-deoxy-5-iodocytidine (dC I ) or from well-known 2′-deoxy-7-iodo-7-deazaadenosine (dA I ), 30 which were triphosphorylated to the corresponding dNTPs (dC I TP and dA I TP). 5 The SuzukiMiyaura cross-coupling of iodinated nucleosides (dC I or dA I )…”

“…2 We and others have previously reported labelling of DNA by diverse redox labels, including ferrocene, 3,4 aminoand nitrophenyl, 5 Os(bpy) 3 -, 6 anthraquinone, 3,7,8 benzofurazane, 9 azidophenyl, 10 methylene-blue, 11,12 polyoxometalates 13 or methoxyphenol 14 through polymerase incorporation of redox-labelled 2′-deoxyribonucleoside triphosphate (dNTP). Combination of several orthogonal redox labels could be used for multipotential redox-coding of DNA nucleobases for development of electrochemical minisequencing techniques.…”

“…The orthogonality requires labelling of each nucleobase by a different redox-active group (having a different redox potential), and each label should be "readable" in the presence of all the other labels and give a ratiometric signal intensity. Our first generations of redox coding 5,7 suffered from weak and overlapping signals, limited stability and difficult incorporation of some labels into DNA. More recently, we reported the first orthogonal and ratiometric set of two reducible labels (nitrophenyl and benzofurazane) useful for electrochemical minisequencing of short DNA stretches.…”