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Proceedings Paper

Force-activated substrates for high-precision, high-throughput optical trapping assays of ssDNA motor proteins (Conference Presentation)
Author(s): Stephen Okoniewski; Thomas T. Perkins

Paper Abstract

Optical-trapping-based assays can measure individual proteins bind to and move along DNA with sub-nm resolution, and have yielded insight into a broad array of protein-DNA interactions. Unfortunately, collecting large numbers of high-resolution traces remains an ongoing challenge. Studying helicase motion along DNA exemplifies this challenge. One major difficulty is that helicase binding often requires a single stranded (ss)-double stranded (ds) DNA junction flanked by ssDNA with a minimum size and orientation. Historically, creating such DNA substrates is inefficient. More problematic is that data throughput is low in standard surface-based assays since all substrates are unwound upon introduction of ATP. The net result is ~2–4 high-resolution traces on a good day. To improve throughput, we sought to turn-on or activate a substrate for a helicase one molecule at a time and thereby sequentially study many molecules on an individual microscope slide. As a first step towards this goal, we engineered a dsDNA that contains two site-specific nicks along the same strand of the dsDNA but no ssDNA. Upon overstretching the DNA (F = 65 pN), the strand between the two nicks was mechanically dissociated. We demonstrated this with two different substrates: one yielding an internal ssDNA region of 1100 nt and the other yielding a 20-bp long hairpin flanked by 30 nt of ssDNA. Unwinding a hairpin yields a 3-fold larger signal while the 30-nt ssDNA serves as the binding site for the helicase. We expect that these force-activated substrates to significantly accelerate high-resolution optical-trapping studies of DNA helicases.

Paper Details

Date Published: 10 November 2016
PDF: 1 pages
Proc. SPIE 9922, Optical Trapping and Optical Micromanipulation XIII, 99220W (10 November 2016); doi: 10.1117/12.2238606
Show Author Affiliations
Stephen Okoniewski, Univ. of Colorado Boulder (United States)
Thomas T. Perkins, Univ. of Colorado Boulder (United States)

Published in SPIE Proceedings Vol. 9922:
Optical Trapping and Optical Micromanipulation XIII
Kishan Dholakia; Gabriel C. Spalding, Editor(s)

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