Highly Conductive Nucleotide Analogue Facilitates Base-Calling in Quantum-Tunneling-Based DNA Sequencing

Abstract

Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient basecalling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides. In this article, we demonstrate that replacement of canonical 2′-deoxyadenosine (dA) with a highly conductive dA analogue, 7-deaza dA, could expand the difference of molecular conductance between four genetic alphabets. Additionally, systematic evaluation of molecular conductance using a series of dA and dG analogues revealed that molecular conductance of the nucleotide is highly dependent on the HOMO level. Thus, the present study demonstrating that signal characteristics of the nucleotide can be modulated based on the HOMO level provides a widely applicable chemical approach and insight for facilitation of single molecular sensing as well as DNA sequencing based on quantum tunneling.

Length Discrimination of Homo-oligomeric Nucleic Acids with Single-molecule Measurement

“…In the previous reports, it is suggested that sample proportional with transmission, is represented as follow equation. [28][29][30][31] (2) Here, ε is the conductive molecular orbital alignment from gold Fermi level, and This inference is consistent with that analysis with current and duration time provides highest accuracy, that is, the single-molecule signal of each homo-oligomer has characteristics of time and current.…”

Combined quantum tunnelling and dielectrophoretic trapping for molecular analysis at ultra-low analyte concentrations

“…This was done by performing chronoamperometric measurements using~1.1 nm gap at a bias voltage of 50 mV. This gap size was chosen as it is slightly larger than the dimensions of a single nucleotide (<1 nm) 16,35 . Each analyte type produced current transients that corresponded to specific peak conductance (ΔG), with dGMP (240 ± 36 nS) > dAMP (180 ± 33 nS) > dCMP (161 ± 5 nS) > dTMP (120 ± 10 nS) (Fig.…”

Chemical‐Labeling‐Assisted Detection of Nucleobase Modifications by Quantum‐Tunneling‐Based Single‐Molecule Sensing

“…Although efficient interpretation of each signal is essential for high‐throughput analysis, similarities in molecular conductance seriously impede the statistical interpretation of signals obtained within a short time frame. As previous works have shown theoretically and experimentally, the molecular conductance is highly correlated with the HOMO energy level of the nucleobase ( ϵ HOMO ), more specifically, the physical parameter 1/( E F − ϵ HOMO ) 2 , in which E F is the Fermi energy of the electrode. This indicates the intrinsic difficulties in identifying electron‐withdrawing or electronically neutral modifications, including oxidative variants of methyl groups, such as hydroxymethyl, formyl, and carboxyl groups (Figure A), which result only in small differences in 1/( E F − ϵ HOMO ) 2 of the nucleotides (Table S1 in the Supporting Information).…”

“…To measure the molecular conductance of BzIm‐dU relative to canonical nucleotides, we synthesized BzIm‐dU flanked by two abasic mimics (Figure A). Previous work showed that dG was the most conductive of the canonical nucleotides . Therefore, if BzIm‐dU is further conductive compared with dG, BzIm‐dU will be efficiently detectable due to the high conductance profile over all canonical nucleotides and a high signal‐to‐noise ratio.…”

“…The resulting conductance histograms did not provide single peak distributions, but could be fitted by two Gaussian curves to provide two conductance peaks, G L and G H , with lower and higher values, respectively (Figure C). Previous work revealed that G H reflected electron transfer mediated by the nucleobase and G L corresponded to electron transfer through less conductive structures, such as ribose . Therefore, we adopted G H as a measure of molecular conductance of the nucleobase.…”