All forms of life require DNA polymerases for the accurate replication and repair of DNA.  Bioconjugating single copies of DNA polymerase to carbon nanocircuits allowed for the electronic monitoring of the enzyme�s dynamics for multiple turnovers (�1000) over multiple minutes (>10).  Analysis of the electronic signal allowed independent analysis of the enzyme�s time spent in the open and closed conformations.  The approach uncovered six independent parameters governing the mechanism of DNA polymerase. For example, the enzyme had faster processing rates for homopolymeric templates of poly(dG) and poly(dC) versus poly(dA) and poly(dT) templates.  In addition, enzymatic processing of homopolymeric poly(dG)/(dC) and poly(dA)/(dT) templates revealed differences in the signal-to-noise ratios of their electronic signal.  Taken together, the results provide new insight into DNA polymerase mechanism and conformational dynamics.  In the future, this approach could allow the engineering of novel DNA polymerase variants with customizable read-out for applications in DNA sequencing.

Single-molecule processing of DNA polymerase I (Klenow fragment) monitored by carbon nanocircuits

245th National Meeting (2013)

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