A toolbox for generating single‐stranded DNA in optical tweezers experiments

Abstract

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.

Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

“…Using two independent optical traps, individual DNA molecules could be manipulated while simultaneously detecting the tension on the DNA. dsDNA molecules were biotinylated on the 3′ ends of both top and bottom DNA strands (Candelli et al., 2013) and were tethered to optically trapped streptavidin-coated polystyrene microspheres (Figure S1D). Single ssDNA tethers (Figure S1E) were generated by biotinylation of both the 5′ and 3′ ends of the top strand of a dsDNA molecule and subsequent detachment of the bottom strand by force-induced melting (Candelli et al., 2013).…”

“…dsDNA molecules were biotinylated on the 3′ ends of both top and bottom DNA strands (Candelli et al., 2013) and were tethered to optically trapped streptavidin-coated polystyrene microspheres (Figure S1D). Single ssDNA tethers (Figure S1E) were generated by biotinylation of both the 5′ and 3′ ends of the top strand of a dsDNA molecule and subsequent detachment of the bottom strand by force-induced melting (Candelli et al., 2013). After incubation of the dsDNA and ssDNA constructs in a GFP-RAD52 solution, the DNA tethers were brought into a buffer channel where DNA-bound proteins could be visualized in the absence of fluid flow and fluorescent proteins in solution (Figures S1F and S1G).…”

Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution

“…To quantify the rate and size of RAD51 nucleation on ssDNA directly, we used force-induced melting to generate the DNA substrate (18). A dsDNA molecule (48.5 or 38.4 kbp) labeled with biotins at the 3′ and 5′ ends of the same strand was captured from both ends with two streptavidincoated microbeads held by independent dual-trap optical tweezers.…”

“…Here we combine quantitative fluorescence microscopy, dualtrap optical tweezers, microfluidics (17), and force-induced DNA melting (18) to quantitatively monitor individual RAD51 nuclei and their growth on both ssDNA and dsDNA with single-monomer sensitivity. These measurements make it possible to directly determine nucleus size, binding rates, and filament growth rates, which may serve as the basis for understanding the function and role of mediators and other factors controlling RAD51 functions.…”

Knotty Problems during Mitosis: Mechanistic Insight into the Processing of Ultrafine DNA Bridges in Anaphase

“…It is possible to generate a section of ssDNA with optical tweezers by stretching a dsDNA molecule to forces beyond 65-70 pN, which induces base pair melting. Occasionally, in the presence of a nick (a break in one of the strands), the melted strand dissociates, yielding a permanent stretch of ssDNA (Candelli et al 2013). A more controlled way to generate a ssDNA/dsDNA hybrid is by using the exonuclease activity of the T7 DNA polymerase induced by putting the DNA under tension (Wuite et al 2000;Hoekstra et al 2017).…”