Extended Data Fig. 2: Design and construction of the nanorotor DNA construct.
From: The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex
a, Scheme of the construct components including four dsDNA fragments: a digoxigenin-labelled surface attachment handle (1), the Cascade target sequence (2), a long spacer to lift the magnetic bead out of the evanescent field (4) and a biotin-modified bead attachment handle (5), as well as the origami rotor arm structure that carries attachment sites (yellow) for the gold nanoparticle (3). b, Analysis by agarose gel electrophoresis of the individual components of the DNA nanorotor construct (lanes 1–5, numbered according to a) as well as the ligation products from all components before (lane F) and after PEG purification (lane P). Lane M contains a DNA size marker whose dsDNA fragment length is given on the left. In lane F, a yellow arrow marks the band containing the full nanorotor DNA construct. Additional dominant ligation products at 1.0–1.5 kbp are caused by the self-ligation of both handles due to the use of an excess of handles with symmetric ligation overhangs. Low molecular weight products (<1.5 kbp) were removed in the subsequent PEG precipitation step. Remaining side-products were removed upon magnetic bead binding and washing prior to the tweezers experiments. Gel electrophoresis was performed as described previously42 and performed for 3 independent preparations.
