Identifying single bases in a DNA oligomer with electron tunnelling

Abstract

It has been proposed that single molecules of DNA could be sequenced by measuring the physical properties of the bases as they pass through a nanopore. Theoretical calculations suggest that electron tunnelling can identify bases in single-stranded DNA without enzymatic processing, and it was recently experimentally shown that tunnelling can sense individual nucleotides and nucleosides. Here, we report that tunnelling electrodes functionalized with recognition reagents can identify a single base flanked by other bases in short DNA oligomers. The residence time of a single base in a recognition junction is on the order of a second, but pulling the DNA through the junction with a force of tens of piconewtons would yield reading speeds of tens of bases per second.

Figure 1

Reading a single base within a heteropolymer. (a) Benzamide groups on the probe and substrate bind bases in the polymer to give a signal dominated by the shortest tunneling path (highlighted for the connection to the single A in d(CCACC)). (b) Characteristic bursts of tunneling noise with large, infrequent spikes signaling C and smaller, more frequent spikes signaling A. (Background tunnel current is 10 pA, bias + 0.5V). The spike labeled * (off-scale at 0.11 nA) is non specific, and rejected from the analysis. (c) Rolling average of the spike height (0.25s window, 0.125 s steps) and spike frequency (d). C bases generate a negligible number of spikes below 0.015 nA (red line). (e) Probability that the signal comes from a A (shown by the red line on panel c) or a C (blue line). For signal amplitudes >0.015 nA, probabilities are calculated as described in the supporting information (values are not normalized to add to 1 in these regions). This burst of signal was chosen to show a clear example of transitions between A and C bases. Longer time traces (Fig. S9a are dominated by signals from A’s which are preferentially trapped in the junction.

Figure 2

Tunneling signals from nucleotides trapped in a functionalized tunnel gap. (a) Proposed hydrogen bonding modes for all four bases. In practice water must play a role because the observed difference between C and ^5meC would not be accounted for by these structures alone. In phosphate buffered saline, but in the absence of analyte, a 20 pS gap (i= 10 pA, V = + 0.5V) gave a signal free of features, except for some AC coupled line-noise pointed by arrows in (b). (c) – (f) Characteristic current spikes produced when nucleotides dAMP, dCMP, d^mCMP and dGMP were introduced (longer signal runs are given in the online supporting information). dTMP gave no signals. (g) – (j) corresponding distribution of pulse heights. Red lines are fits to two Gaussian distributions in the logarithm of current. (k) Definition of the parameters used to characterize the tunneling signals. Spikes are counted if they exceed a threshold equal to 1.5 × the standard deviation of the noise on the local background. The signals occur in bursts (duration T_B, frequency f_B) each containing current spikes at a frequency f_S. The spikes stay high for a period t_on and low for a period t_off. The total count rate (inset in g–j) is the number of spikes in all bursts divided by the measurement time.

Figure 3

Tunneling signal distributions from oligomers resemble those of the constituent nucleotides. (a, c, e) Representative current traces from d(A)₅, d(C)₅ and d(^mC)₅ with the corresponding distributions shown in b, d and f. Red lines are fits with parameters similar to those used for nucleotides (online supporting information). The black lines are the fits to the corresponding nucleotide distributions shown in Figs. 2 g,h and i. “HCT” labels some of the high current features seen in homopolymers but not nucleotides (b and f). (g and i) Current traces from mixed oligomers, d(ACACA) and d(C^mCC ^mCC), with corresponding current distributions (h and j). Red lines are scaled homopolymer fits, with the green-dashed line showing the “C” contribution, the orange-dashed line showing the “A” contribution and the purple-dashed line the ^mC contribution. The data are well described by the homopolymer parameters though some intermediate signals (“1”) and new high current features (2) show that the sequence context affects the reads a little. The colored bars on the current traces mark bursts of A-like signals (orange), C-like signals (green) and ^mC-like signals (purple).

Figure 4

The lifetime of the reading complex is on the order of a second at zero force. (a) AFM gap functionalization where the blue line represents a 34 nm PEG linker. (b) representative force curves showing: (i) Pulling on more than one molecule at a time – the force baseline is not restored after each break, and the z-extension (corrected for tip displacement) is > 34 nm. (ii) A single molecule curve of the type accepted by the software. The force returns to the baseline after the bond breaks and the corrected extension is ~ 34 nm. (c) Histograms of bond breaking forces at the pulling speeds marked. The solid lines are maximum likelihood fits to the heterogenous bond model. (d) Bond survival probability plotted versus bond breaking force for the four pulling speeds, fitted by the same heterogeneous bond model parameters (solid lines). These fits yield a zero-force off rate of 0.28 s⁻¹ implying that the assembly lives for times on the order of seconds in a nanogap, much longer than the lifetime in solution. For details see ref. .