Fig. 2. Biochemical characterization of the LdCsm effector complex.

a Schematic of three different homologous target RNAs: CTR cognate target RNA carrying 6-nt 3ʹ anti-tag with mismatch to the 5ʹ tag of the corresponding crRNA, NTR noncognate target RNA containing 8-nt 3ʹ anti-tag that is complementary to the 5ʹ tag of the corresponding crRNA, PTR 40 nt protospacer target RNA completely lacking 3ʹ anti-tag. b Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. c Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10–60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. d Analysis of cOA synthesis by LdCsm. Approximately 2 nM [α-³²P]-ATP was mixed with a range of cold ATP (48 nM–1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min; the S. islandicus Cmr-α complex was used as the positive reference.