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Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2016)

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.

    Cover page: 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
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2017)
    DNA polymerases must discriminate the correct Watson-Crick base pair-forming deoxynucleoside triphosphate (dNTP) substrate from three other dNTPs and additional triphosphates found in the cell. The rarity of misincorporations in vivo, then, belies the high tolerance for dNTP analogs observed in vitro. Advances over the last 10 years in single-molecule fluorescence and electronic detection of dNTP analog incorporation enable exploration of the mechanism and limits to base discrimination by DNA polymerases. Such studies reveal transient motions of DNA polymerase during substrate recognition and mutagenesis in the context of erroneous dNTP incorporation that can lead to evolution and genetic disease. Further improvements in time resolution and noise reduction of single-molecule studies will uncover deeper mechanistic understanding of this critical, first step in evolution.
      Creative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2017)
      DNA polymerases must discriminate the correct Watson-Crick base pair-forming deoxynucleoside triphosphate (dNTP) substrate from three other dNTPs and additional triphosphates found in the cell. The rarity of misincorporations in vivo, then, belies the high tolerance for dNTP analogs observed in vitro. Advances over the last 10 years in single-molecule fluorescence and electronic detection of dNTP analog incorporation enable exploration of the mechanism and limits to base discrimination by DNA polymerases. Such studies reveal transient motions of DNA polymerase during substrate recognition and mutagenesis in the context of erroneous dNTP incorporation that can lead to evolution and genetic disease. Further improvements in time resolution and noise reduction of single-molecule studies will uncover deeper mechanistic understanding of this critical, first step in evolution.
        Cover page: Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2015)
        DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.
          Cover page: Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) NanocircuitsCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2016)
          As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.
            Cover page: Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA LengthsCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2016)
            As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.
              Cover page: Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2015)
              Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.
              • 1 supplemental PDF
              Cover page: Shear‐Stress‐Mediated Refolding of Proteins from Aggregates and Inclusion BodiesCreative Commons 'BY' version 4.0 license
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