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. 2018 Mar 12;115(13):3368–3373. doi: 10.1073/pnas.1717725115

Fig. 3.

Fig. 3.

MpAgo RNP programmed with 5′-BrdU gRNA binds ssRNA substrates with high affinity. (A) Cartoon of three duplexes of the gRNA and different ssRNA substrates with varying degrees of complementarity to the gRNA used in binding assays: full 21-nt complementarity (Top, red), 18-nt complementarity with three mismatches at the 3′-end of the gRNA (Middle, orange), and 15-nt complementarity with six mismatches at the 3′-end of the gRNA (Bottom, black). The first nt at the 5′-end of the gRNA does not base pair because of MpAgo interactions (Fig. 2A). (B) Filter-binding assays with 21-nt complementarity (red), 18-nt complementarity (orange), or 15-nt complementarity (black) in the presence of 10 μg/mL heparin. The amount of bound ssRNA substrate is plotted as a function of MpAgo RNP concentration. The obtained average Kds for MpAgo RNP and the substrates with 21-, 18-, and 15-nt complementarity are 500 ± 115 pM, 107 ± 6 pM, and 83 ± 8 pM, respectively. Data are represented as the mean ± SD from three independent experiments.