Revealing the Dynamics of Single-Molecule Reactions in a Single-Molecule Nanoreactor

Abstract

The dynamics of bond breaking and formation at the single-molecule level are of both fundamental importance and practical significance. Tremendous efforts have been made over the last decade toward the visualization of such transient phenomenon, with quite a few amazing single-molecule techniques being designed and developed. However, it remains a major challenge to realize the truly single-molecule (cascade) reactions largely due to the stochastic nature of, e.g. molecular motions. Inspired by the enzymatic biosynthesis, the authors consider that a nanoscale confined space, or nanoreactor, must be essential to the highly ordered single-molecule reactions. Moreover, an ideal nanoreactor also needs to possess a well defined structure that is of comparable size to the individual reactant molecules. Hence, the rationally-mutated biological nanopores may be applied as a new brand of single-molecule nanoreactors, where the entry of reactants and exit of products are voltage-driven. Herein, a mutant areolysin was adopted to study the unique confinement induced selectivity of nanopore reactor. In addition to regio- or stereo-selectivity, it is hypothesized that the mismatch between the bond formation kinetics of reactant molecules and their translocation speeds can offer an extra level of control on reaction selectivity. To prove this concept, the reaction dynamics of three constitutional isomers, 2-, 3-, and 4-mercaptobenzoic acid, at seven identical reaction sites (i.e. at the same position on each monomer) were examined in this work. Surprisingly, those tiny structural variations of three isomers could be easily distinguished by not only the different current blockades caused by each tethered molecule, but also the most frequent and prolonged current levels as well. This observation shed light on the transient dynamics of single-molecule reactions within a single-molecule nanoreactor, providing a crucial first step to the ultimate objective of 100% yield of targeted product.