Measurement of single-molecule reactions provides insight into their microscopic mechanism, which is often hidden in ensemble measurements. However, the stochastic nature of single molecule reactions can obscure information when short-term measurements are performed. Herein, hours-long measurements of a single molecule are reported, in which single-molecule reactions are observed. A single DNA duplex containing a C:C base pair mismatch is captured and held for hours in the channel of wild-type α-hemolysin, which acts as a nanoreactor. Fluctuations in the ionic current through the protein channel allow us to monitor the kinetics of a nucleotide in the C:C base flipping from an intra- to an extra-helical state.[1] The rates for base pair flip-in (kin) and flip-out (kout) are on the orders of 10-2 s-1 to 10-1 s-1 and are stable over the measurement (hours). A protonation/deprotonation reaction is involved in the base flipping dynamics, as evidenced by pH-dependent kinetics and kinetic isotope effect. A kinetic model is proposed to explain the pH-dependent dynamics of C:C mismatch, and the protonation reactions is deduced to occur at a rate near diffusion control. To our knowledge, these are the longest single-molecule measurements performed using a nanopore and show its great promise as a nanoreactor.

α-Hemolysin as a nanoreactor for hours-long monitoring of single molecule reactions

254th National Meeting (2017)

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