Publication protocol
"Oligonucleotides L1, L2, TS1 and TS2 were synthesized by Trilink and resuspended in formamide (Merck) at 37 °C, so as to obtain a 10 μg μl–1 final concentration. Typically, ~100 μg of each Li was combined with an equimolar amount of the associated TSi and incubated for 12 h at 25 °C to yield the corresponding Ji product by click-chemistry coupling (Step a1 in Supplementary Fig. 1). Both J1 and J2 branched oligonucleotides were then purified by electrophoresis on an 8 M urea and 8% acrylamide/bisacrylamide (29:1) gel, as previously described17. Subsequently, J1 and J2 were used as primers in the PCR amplification of a ~2.0 kbp fragment of the Charomid 9-5 ΔSbfI template with the Expand High Fidelity polymerase system (Roche) (Step a2 in Supplementary Fig. 1)—the template used is a derivative of the Charomid 9-5 plasmid in which the native SbfI site was removed by SbfI digestion, fill-in and ligation with the Quick Blunting kit (New England Biolabs). The resulting branched PCR products, which are the precursors of the tips and shanks of the J-DNA tweezers, were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).
In parallel, the leash precursor was synthesized as follow. Its ~630 bp backbone was amplified by PCR using the Platinum Pfx DNA polymerase (Invitrogen), the Charomid 9-5 ΔSbfI template and the Charo-3600-MluI and Charo-4230-SbfI primers. The blunt-end PCR product was then digested at one end with SbfI and cloned into the pUC18 vector using the SbfI and SmaI restriction sites. The resulting DNA plasmid, 690_pUC18, was amplified and purified using the NucleoBond Xtra maxiprep kit (Macherey-Nagel), and digested with the MluI and SbfI restriction enzymes. The digestion products were separated using electrophoresis on a 1% agarose gel and extracted using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).
Next, the L1 and L2 oligonucleotides of the branched PCR product, which did not participate in the amplification reaction, were annealed in an equimolar ratio to the O1-Comp and O2-Comp oligonucleotides, respectively (Step a3 in Supplementary Fig. 1). This reaction took place at a final concentration of ~270 nM in each strand for 1 h at room temperature in 1× CutSmart buffer (New England Biolabs). This assembly was subsequently combined with the leash precursor at a ~130 nM final concentration, each in 1× CutSmart buffer, along with 10 units (U) of SbfI-HF, 10 U of MluI-HF, 10 U of NsiI-HF, 5 U of AscI, 1,000 U of T4 DNA ligase (New England Biolabs), 1 mM ATP and 1 mM DTT (Step a4 in Supplementary Fig. 1). After overnight incubation at 25 °C, the mixture was heat inactivated at 65 °C for 20 min. Then, in a one-pot reaction that took place at 37 °C for 8–16 h, the tips were nicked with 30 U of Nb.BvCI (New England Biolabs) and the extremities of the shanks were digested with 100 U of XbaI and 50 U of SacI (New England Biolabs) (Step a5 in Supplementary Fig. 1). The product was separated on a 1.5% agarose gel at 37 °C and extracted using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).
Finally, the ~2.7 kbp construct obtained above was ligated at the SacI site to a ~1 kbp dsDNA fragment multiply labelled with biotin and at the XbaI site to a ~1 kbp dsDNA fragment multiply labelled with digoxigenin (Step a6 in Supplementary Fig. 1). These labelled DNA fragments were synthesized by PCR as previously described36,56 in the presence of dUTP-biotin and dUTP-digoxigenin (Roche), respectively. The ligation reaction was conducted for 3 h at room temperature in 10 μl of 1× T4 DNA ligase buffer (New England Biolabs) that contained the construct at 3 nM, biotin and digoxygenin-labelled ~1 kbp fragments at 10 nM each, and 200 U of T4 DNA ligase. The enzyme was then heat inactivated and the resulting J-DNA tweezers were aliquoted and stored at −20 °C."
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