Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.

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Cytosine-rich nucleic acid sequences found in human DNA can adopt multiple intramolecular structures identified as i-motifs that are dependent on physiochemical solution conditions. The focus of this study is a four-stranded structure from the promoter of the human c-MYC gene. This compact, stable, and monomeric structure forms upon a decrease in pH (<5.0) causing hemiprotonation of a cytosine that results in a stable C-Cþ hydrogen bond. Sedimentation velocity experiments were performed with the analytical ultracentrifuge in order to determine the hydrodynamic properties of the folded i-motif. The sedimentation velocity experiments were carried out in buffer conditions that differed in pH (4.5-8.0), salt type (NaCl and KCl), and salt concentration (up to 400 mM). Experiments were run at different pH values in order to observe the linkage between the pKa of the Hoogsteen base pair and i-motif folding. High salt concentration was used to avoid non-ideality (primary charge effect) observed when nucleic acids sediment at low salt concentration. The data indicates that the S20,w value increases when the primary charge effect is overcome at a higher salt concentration. The experimental S20,w values are compared with values obtained by bead model simulations using SOMO as implemented in Ultrascan 3. (Supported by UMC AUC Facility.