Real-Time Recording the Dynamic Catalytic Heterogeneity of Enzymatic Reactions Using a Nanopore

Enzymatic reactions in cells control the diversity of biomolecular composition, structure, and function, by virtue of their dynamics and heterogeneity. Here, we describe the use of a protein nanopore to monitor, in real time, the action of Exonuclease I (Exo I) on its substrate (homogeneous and heterogeneous short single-stranded DNA, ssDNA) on a single-reactant molecule basis. The nanopore-based single-molecule measurement, combined with a transition kinetic analysis, determines the temporal dynamics and heterogeneous cleavage and release pathways of ssDNA by Exo I. The results demonstrate a stepwise cleavage that is sequence-dependent on short ssDNA molecules (<15 nt), which differs from the kinetic model based on bulk measurements. In addition, we show that damaged DNA irreversibly changes the enzymatic reaction processes by Exo I. Thus, nanopores might prove to be useful for studying multienzyme cascade reactions at the single-molecule level.