Extended Data Fig. 2: Analysis of the optimal reaction conditions for NbaSPARDA in vitro.
a, Structure of gRNA and tDNAs (single-stranded or double-stranded) used in the assays. b, Activity of SPARDA with single-stranded tDNAs of various lengths (20, 30, and 50 nt), in the absence and in the presence of gRNA. c, Activity of SPARDA with double-stranded (ds) tDNA (20 nt, lanes 3–5, or 50 nt, lanes 6–8), tested for 60 minutes at 30 °C or 40 °C. Control reactions were performed with single-stranded (ss) tDNA. The non-target DNA strand was annealed with the complementary target strand at the 1:1 or 3:2 ratio. While low level of SPARDA activity was observed at the 1:1 ratio (lanes 3 and 8), it was suppressed at the 3:2 ratio (lanes 4–5 and 6–7), indicating that this activation resulted from the presence of non-annealed single-stranded tDNA. The position of non-denatured 50 nt duplex DNA is indicated with an asterisk. d, Activity of SPARDA in buffers containing various divalent cations (5 mM each), with either 5 mM or 100 mM KCl. d, Activity of SPARDA with different concentrations of Mg2+ (left, with 5 mM KCl) or Mn2+ (right, with 100 mM KCl). f, Activity of SPARDA with different concentrations of Mn2+ in the absence and in the presence of 5 mM Mg2+ (with 100 mM KCl). g, Activity of SPARDA at various pH. h, Activity of SPARDA at various temperatures. i, Sequences of gRNAs and tDNAs used for analysis of the optimal gRNA structure. For gRNAs with substitutions of the first or second nucleotides, tDNA contained complementary substitutions at corresponding positions (indicated with ‘N’). j, Activity of SPARDA with gRNAs of different lengths. k, Kinetics of substrate DNA cleavage with 5’-phosphorylated and 5’-OH gRNAs. l, Effects of substitutions of the 5’-nucleotide in gRNA (lanes 2–6) or in gDNA (lanes 7–10) on the activity of SPARDA. SPARDA is activated only by gRNAs with 5’-A (lanes 2 and 3). m, Effects of substitutions of the second nucleotide in gRNA on the activity of SPARDA. The 5’-end dinucleotides in each gRNA are shown above the gel. All reactions were performed at 30 °C in reaction buffers containing 5 mM Mn2+ (with 100 mM KCl) or 5 mM Mg2+ (with 5 mM KCl), unless otherwise indicated. The reactions in panels b, c, k, j, l were performed in the presence of Mn2+. All reactions except for panels c and h were performed at 30 °C. In each panel, representative gels from several independent experiments are shown, which produced similar results: 4 replicates in panel b, 2 replicates in panels c-m. Positions of gRNA, tDNA, single-stranded collateral substrate DNA (71 nt) and the cleavage products are indicated. The lengths of guides, targets and substrates are shown in nucleotides (see Supplementary Table 1 for oligonucleotide sequences).
