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Abstract
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.
Highlights
In populations of organisms, cells, or molecules, atypical individuals can exert disproportionate roles
Under the direction of a single-stranded DNA template, the polymerase incorporates dNTPs into a complementary template strand
The nascent strand lengthens base-by-base as the polymerase accepts an incoming dNTP and catalyzes its addition to the new strand’s 31 -hydroxyl terminus [74]
Summary
Cells, or molecules, atypical individuals can exert disproportionate roles. In the least consequential cases, these individuals merely nudge the average activity or phenotype of a population; but in more severe cases, they turn on entirely new responses or enable pathways with pathological consequences [1]. At an even finer scale, a single errant or mutant biomolecule can disrupt a cell signaling pathway or inappropriately activate transcription [5,6]. Understanding cause and effect in a population is very difficult when single individuals can change the behavior of the overall system, and addressing this challenge requires a detailed study of protein function at the individual level. Over the past two decades, tremendous progress has been made developing techniques that can address the challenge of characterizing the activity of single molecules.
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