Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers

Abstract

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2,000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.