Fig. 4: HrAgo1 displays a unique hybrid mode of guide organization and target binding.

a Close-up view of guide RNA organization by HrAgo1. b Comparison of structural features involved in guide RNA seed segment organization in HrAgo1, EfPiwi, and hAgo2. c Schematic of the single-molecule binding assay. Only when the HrAgo1-guide complex binds to the target, FRET will occur. d Schematic representation of a guide and target used in the single-molecule binding assay. Complementary nucleotides are indicated in dark gray and mismatched nucleotides are shown in light gray. N6 indicates base pairing with nucleotides 2–7 of the guide. e A representative time trace with four binding events, of which the dwell time (Δτ) of one is indicated. f Dwell time distributions for N4, N6, and N15. N4 and N6 are best fit with a single and double exponential, respectively. N15 cannot be fit and shows stable binding. The distributions and fits for the other match lengths and representative time traces can be found in Fig. S6. g Bubble plots showing the increase of dwell times for increasing complementarity between the guide and target for HrAgo1, EfPiwi and hAgo2. The area of the bubbles corresponds to the percentage of the total population belonging to this sub-population. The dashed lines indicate the time resolution (0.1 s) and the observation time limit (200 s). In the bubbles, the mean ± SD are indicated, and the darker shaded area indicates the SD of the fractional values of at least three independent experiments. The dwell times for EfPiwi were obtained in a similar way as for HrAgo1. For hAgo2, previously published dwell times were used⁴¹. Source data are provided in the Source data file.