Extended Data Fig. 4: Activities of NbaSPARDA and CmeSPARDA with various collateral substrates.
a, Kinetics of cleavage of supercoiled plasmid DNA (pBAD) by activated NbaSPARDA. Control reactions were performed for 40 minutes without SPARDA (lane 1) or without gRNA (lane 2). b, Cleavage of linear dsDNA fragments of indicated lengths (obtained after treatment of pBAD by restriction endonucleases with gel purification). c-e, Analysis of collateral cleavage of 55 nt ssRNA, RNA/DNA duplex and dsRNA. The reactions were performed with 5’-FAM and 5’-HEX labeled substrate RNA and DNA oligonucleotides, as indicated below the gels. f, Analysis of gRNA and tDNA cleavage by NbaSPARDA in the absence of collateral substrates. g,h, Comparison of the activities of wild-type (WT) NbaSPARDA and its MID mutant (g) or CD (h) mutants with the collateral ssDNA substrate (71 nt) in the presence of Mg2+ and Mn2+. i, Cleavage of the ssDNA substrate (71 nt) by CmeSPARDA. j, Cleavage of FAM-labeled 55 nt ssRNA substrate by CmeSPARDA. k, Cleavage of plasmid DNA (pBAD) and its linear dsDNA fragment (~3000 bp) in the presence of gRNA and tDNA. Linear plasmid (lane 3) was obtained by treatment with HindIII. Control reactions in lanes 4 and 7 were performed in the absence of gRNA. M, length marker. Linear DNA fragments in panels b and k have lower gel mobility in comparison with molecular weight markers (lane 1) than expected, likely due to differences in the loading buffer composition (see Materials and Methods). The lengths of guides, targets and substrates in panels c-j are shown in nucleotides (see Supplementary Table 1 for oligonucleotide sequences). The reactions were performed for 30 min (1 hour in panel i) at 30 °C with standard 20 nt gRNA (20 nt 5’-A-RNA) and 50 nt single-stranded tDNA (200 nM each; 100 nM in panels a,b,g,h). The samples contained 200 nM NbaSPARDA (panels a,b), 500 nM NbaSPARDA (panels c-h) or 350 nM CmeSPARDA (panels i-k). The experiments in panels a,b,g and h were performed with WT SPARDA samples containing admixture of endogenous gRNAs, so that the relative activity of guide-free WT SPARDA would be even higher in these assays (see below Extended Data Fig. 8a, c). The reaction buffers contained 5 mM Mg2+ (with 5 mM KCl) or 5 mM Mn2+ (100 mM KCl), as indicated. The reactions in panels a,b and k were performed in buffers containing 5 mM Mg2+ (with 5 mM KCl). In all cases except panel j, nucleic acids were visualized by SYBR Gold staining; 5’-FAM-labeled RNA in panel j was visualized by fluorescence scanning. In each panel, representative gels from several independent experiments are shown, which produced similar results: 3 replicates in panels a,c,g,h,i,j,k; 2 replicates in panels b,d,e,f. Positions of gRNA, tDNA, collateral substrates and the cleavage products are indicated. See Supplementary Table 1 for oligonucleotide sequences.
