Extended Data Fig. 3: Multiple-round DNA cleavage by NbaSPARDA.

a-c, Cleavage of ssDNA substrates. a, Cleavage of an excess of 5’-P³²-labelled 71 nt tDNA (5 μM) with increasing amounts of the activated SPARDA complex (from 10 to 500 nM, SPARDA:gRNA:tDNA = 1:1:1). b, Multiple-round cleavage of the 71 nt tDNA substrate (5 μM) by activated NbaSPARDA (500 nM SPARDA, 500 nM gRNA, 500 nM tDNA). c, Multiple-round cleavage of 5’-P³²-labeled 55 nt tDNA (5 μM) under the same conditions. d-e, Cleavage of a ssRNA substrate. d, Cleavage of 5’-FAM-labeled 55 nt ssRNA (5 μM) with increasing amounts of the activated SPARDA complex (from 10 to 500 nM, SPARDA:gRNA:tDNA=1:1:1). e, Multiple-round cleavage of the 55 nt RNA substrate (5 μM) by activated NbaSPARDA (500 nM SPARDA, 500 nM gRNA, 500 nM tDNA). All reactions were performed in three replicates at 30 °C in the buffer containing 5 mM Mn²⁺ and 100 mM KCl. The reaction products were visualized by phosphorimaging (panels a-c) and fluorescence scanning (panels d-e). In each case, representative gels from three independent experiments are shown. f, Kinetics of ssDNA and ssRNA cleavage by NbaSPARDA. Means and standard deviations from three replicates. See Supplementary Table 1 for oligonucleotide sequences.

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