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Detection of DNA Polymerase I (Klenow Fragment) Activity Using Single-Molecule Electronic Nanocircuits and Toward the In Vitro Evolution of High-Affinity Binding Partners for Membrane Proteins From Phage-Displayed Affinity Reagent Libraries

  • Author(s): Pugliese, Kaitlin
  • Advisor(s): Weiss, Gregory A
  • et al.
Abstract

DNA polymerases are central biocatalysts in the life cycle of all organisms. Structural transitions of this enzyme during the incorporation of deoxynucleoside triphosphate (dNTP) substrates govern the accurate replication of DNA. Biophysical tools have emerged as useful techniques for understanding these structural transitions, and are continually being improved. Electronic-based techniques, in particular, provide many advantages over other biophysical techniques, including long duration monitoring and enhanced time resolution. For example, recently developed single-walled carbon nanotube field-effect transistors that measure DNA polymerase I Klenow Fragment (KF) activity serve as a valuable method for label-free detection of biocatalysis. In my Ph.D. work, I have sought to develop this technique further to better understand enzyme activity and take advantage of our observations for biotechnological applications.

Incorporation of dNTP analogs with KF-functionalized nanocircuits challenged the molecular recognition of these substrates by KF. For some dNTP analogs, an alternative motion of KF during dNTP incorporation was observed and may be valuable in the discrimination of DNA bases in a template. The nanocircuits also accurately identified DNA template lengths when such templates were composed of highly repetitive sequences. Accumulation of several data sets further enabled precise measurement of DNA template lengths. Initial experiments of dNTP incorporation with a KF nanocircuit measured at high bandwidths demonstrated that the accurate measurement of DNA template lengths required the lower time resolution. The higher bandwidth experiments revealed previously unobserved KF motions that lasted for short durations, making this technique unsuitable for counting DNA bases. However, improvement to KF nanocircuits measured at high bandwidth can provide a powerful tool for understanding transient KF dynamics.

To better understand and improve KF nanocircuits, a variety of KF variants were designed and generated. Many of these variants exhibited polymerase activity at the single-molecule level, and are in various stages of progression for their measurement with polymerase-functionalized nanocircuits. The experiments necessary to determine their viability in improving and understanding the electronic signals generated by KF were outlined for future Weiss lab members.

Membrane proteins are a class of biomolecules that also benefit from the improved understanding of their molecular mechanisms. In vitro evolution of high affinity binding partners with stable, rigid phage-displayed protein libraries were attempted for the Helicobacter pylori inner membrane protein Ure-I, but were unsuccessful due to Ure-I’s rapid degradation. An alternative method for in vitro evolution for high affinity binding partners to the Shigella dysenteriae outer membrane protein ShuA was optimized to maintain ShuA in a native membrane environment.

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