Extended Data Fig. 5: Quantification of RNA binding with RBD#3L, RBD#4L, and WT LwaCas13a.
From: Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection
a and c, Representative EMSA gel images of inactive HEPN2 mutants (R1046A/H1051A), that is, dRBD#3L, dRBD#4L, and dWT complexed with crRNA binding to 5 nM of body-radiolabeled (32P) target RNA. (a, T26; c, T1). The protein:crRNA complex with a molar ratio of 1:0.95 was serially diluted to 288, 192, 128, 86, 57, 38, 25, 17, 12, 8 and 5 nM. The 0-nM control was target RNA with reaction buffer. b and d, Calculation of binding affinity between target RNA and RNP. Bound and unbound fractions from a and c were quantified by densitometry and fitted to standard binding isoforms. Mean from two technical replicates was plotted. e, Representative EMSA gel images of protein:crRNA:target ternary complex binding with 100 nM of 5'-FAM-labeled U20 reporter. The dRBD#3L, dRBD#4L and dWT complex with crRNA with a molar ratio of 2:1 was serially diluted to 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.4, 0.2, 0.1 and 0.05 μM. An equal amount of 50 pM non-labeled target (T1) was added to the RNPs to mimic the target-bound ternary complex. The 0-μM control was the reporter with reaction buffer. f, Calculation of binding affinity between reporter RNA and target-bound RNP. Bound and unbound fractions from e were quantified by densitometry and fitted to standard binding isoforms. Mean from two technical replicates was plotted. Means of the dissociation constant with 95% CI are listed in Supplementary Table 3.
