Single-molecule biophysics and techniques
(A) Description of a magnetic tweezers apparatus. A pair of permanent magnets are disposed above a flow chamber that contains nucleic acids tethered magnetic beads. We represent two types of tether, a double-stranded (ds) nucleic acid (left) or a single-stranded (ss) nucleic acids folded on itself, i.e. a hairpin (right). The beads are illuminated with a collimated monochromatic light source and imaged with an oil immersion objective onto a CMOS camera. The 3D position of each bead is determined in real-time using a custom software that is running on a dedicated computer. (B) Characteristic field of view of our high-throughput magnetic tweezers apparatus, which contains hundreds of tethered beads. (C) Step motions stuck reference beads when moving the piezo stage holding the objective by 0.3 nm steps.
Nucleic acids from all organisms are processed by different type of enzymes, such as polymerases and helicases (7). Polymerases belong to a family of enzymes responsible for the replication and the transcription of nucleic acids in all organisms. Single-molecule techniques have shown in the early 90s that the activity of these enzymes is interrupted by a variety of pauses with different biochemical origins. For example, magnetic tweezers have been used to study viral RNA-dependant RNA polymerase (RdRp) to explore the mechanism of mismatch nucleotide incorporation (Figure 2A) (3), a particularly rare event (~1 every 3000 incorporations in error-prone RNA viruses), or long non-catalytic pauses related to backtracking (8). High-throughput magnetic tweezers provides a unique tool to study quantitatively error incorporation (Figure 2B). Using a slightly different approach, where a hairpin replaces the double stranded nucleic acid to tether the magnetic bead, one is able to observe the unwinding activity of a replicative hexameric helicase (Figure 2C) (9).
(A) A dsRNA attaches a magnetic bead to the surface. One strand has a free 3’-end that is used by the polymerase (Pol.) to initiate transcription. Following successful initiation, the polymerase activity converts the dsRNA tether into ssRNA. This activity changes the distance from the bead to the surface by ∆. (B) Transcription activity by P2 polymerase versus time monitored using parallelized detection, yielding 52 traces in a single experiment. (C) A hairpin attaches the magnetic bead to the surface. Preceding the stem of the nucleic acid hairpin, a single-stranded region is used as a loading site for the enzyme of interest, here a hexameric helicase (Hel.). After binding on the loading site, the helicase unwinds the stem of the hairpin, increasing the distance from the bead to the surface by ∆. Both experiments in (A) and (C) rely on the change in extension of the tether to demonstrate enzymatic activity.
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