Force-activated substrates for high-precision, high-throughput optical trapping assays of ssDNA motor proteins
(Conference Presentation)

Stephen Okoniewski; Thomas T. Perkins

doi:10.1117/12.2238606

10 November 2016 Force-activated substrates for high-precision, high-throughput optical trapping assays of ssDNA motor proteins (Conference Presentation)

Stephen Okoniewski, Thomas T. Perkins

Author Affiliations +

Proceedings Volume 9922, Optical Trapping and Optical Micromanipulation XIII; 99220W (2016) https://doi.org/10.1117/12.2238606
Event: SPIE Nanoscience + Engineering, 2016, San Diego, California, United States

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.

Conference Presentation

Citation Download Citation

Stephen Okoniewski and Thomas T. Perkins "Force-activated substrates for high-precision, high-throughput optical trapping assays of ssDNA motor proteins (Conference Presentation)", Proc. SPIE 9922, Optical Trapping and Optical Micromanipulation XIII, 99220W (10 November 2016); https://doi.org/10.1117/12.2238606

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KEYWORDS

Proteins

Optical tweezers

Molecules

Acquisition tracking and pointing

Microscopes

Optical testing

Current controlled current source

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