Single-molecule DNA nanomanipulation: detection of promoter-unwinding events by RNA polymerase.

This article describes a nanomanipulation technique that makes it possible to mechanically and quantitatively stretch and supercoil a single linear DNA molecule. We show how this technique can be extended to the study of protein–DNA interactions that lead to DNA untwisting, particularly to the study of promoter unwinding by RNA polymerase during the initiation of transcription. In our system, a linear 4-kb DNA molecule containing a single promoter is anchored at one end to a treated glass surface and at the other end to a small (1 m) magnetic bead (Fig. 1A). Using a magnetic manipulator, the bead is pulled on and rotated, resulting in supercoiling of the DNA (one negative supercoil per each clockwise rotation; one positive supercoil for each counterclockwise rotation). The three-dimensional position of the bead is determined in real time using videomicroscopy and software analysis, yielding the DNA end-to-end extension, l. Changes in l as a function of force, F, and supercoiling, n, are calibrated. The system is used to observe, in real time, protein–DNA interactions that affect supercoiling. For example, when RNA polymerase is introduced and allowed to bind to and unwind promoter DNA, the corresponding changes in DNA supercoiling and end-to-end extension can be observed by monitoring the bead position in real time. The system is prepared as follows. One end of the 4-kb DNA fragment containing a single promoter is ligated to a 1-kb multiply biotin-labeled DNA fragment, and the other end is ligated to a 1-kb multiply digoxigeninlabeled DNA fragment. Reaction of the resulting DNA fragment with a streptavidin-coated magnetic bead results in attachment of the DNA fragment to the bead, through multiple linkages, to the biotin-labeled end. Deposition of the resulting bead-attached DNA fragment onto an