These authors contributed equally to the work.
A toolbox for generating single-stranded DNA in optical tweezers experiments
Version of Record online: 22 JUN 2013
Copyright © 2013 Wiley Periodicals, Inc.
Volume 99, Issue 9, pages 611–620, September 2013
How to Cite
Candelli, A., Hoekstra, T. P., Farge, G., Gross, P., Peterman, E. J. G. and Wuite, G. J. L. (2013), A toolbox for generating single-stranded DNA in optical tweezers experiments. Biopolymers, 99: 611–620. doi: 10.1002/bip.22225
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at firstname.lastname@example.org
- Issue online: 22 JUN 2013
- Version of Record online: 22 JUN 2013
- Accepted manuscript online: 27 FEB 2013 04:26AM EST
- Manuscript Accepted: 19 FEB 2013
- Manuscript Received: 20 DEC 2012
- Netherlands Organisation for Scientific Research (NWO)
- the Foundation for Fundamental Research on Matter (FOM)
- Advanced Training in Laser Sciences, a Marie Curie Early Stage Training project
- optical tweezers;
- ssDNA–protein interaction;
Essential genomic transactions such as DNA-damage repair and DNA replication take place on single-stranded DNA (ssDNA) or require specific single-stranded/double-stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single-molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single-molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force-induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force-induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.