Constructing a magnetic tweezers to monitor RNA translocation at the single-molecule level.

ABSTRACT

Single-molecule methods have become an invaluable tool in the investigation of the mechanisms of nucleic-acid motors. Magnetic tweezers is a single-molecule manipulation technique that permits the real-time measurement of enzyme activities on single nucleic-acid molecules at high-resolution, high-throughput, and inherently constant force. Here, we describe several aspects of the implementation of magnetic tweezers, with special emphasis on the construction of a simple magnetic trap and, in particular, on the detailed description of image analysis methods to measure the extension changes in nucleic-acid molecules induced by protein activity. Finally, we carefully describe the steps involved in performing a full magnetic tweezers experiment.