Fig. 2: RNase H1 is a 3′-to-5′ exoribonuclease.

a The cartoons on the left illustrate the experimental design in examining the direction of RNase H1 during exonucleolytic cleavage. The directionality of RNase H1 can be distinguished using two microspheres that differ in size. On the right, a representative kymograph shows the directional movements of two RNase H1-Cy3 proteins toward the smaller bead. The corresponding length of the examined molecule is shown below the kymograph. b The hybrid cleavage rate in the presence of 5 nM RNase H1 under 10 pN with a Gaussian fitting in red (n = 21). c The hybrid cleavage processivity in the presence of 5 nM RNase H1 under 10 pN (degrading events, n = 21). Error bars represent SEM. d The experimental design of the smFRET assay depicts that RNase H1-mediated 3′-to-5′ RNA degradation within the 3′-RDH substrate increases the FRET efficiency. The E histograms of the 3′-RDH substrate upon replacing Ca²⁺ with Mg²⁺ for 0 s (black), 100 s (purple), and 200 s (blue) are shown. e The experimental design of the smFRET assay illustrates that RNase H1-mediated 3′-to-5′ degradation of the RNA within the 5′-RDH substrate detaches it from the glass surface. The E histograms of the 5′-RDH substrate upon replacing Ca²⁺ with Mg²⁺ for 0 s (black), 100 s (purple), and 200 s (blue) are shown. f Representative images displaying the fluorescence signals of the 3′-RDH or 5′-RDH substrate after initiating the RNA degradation for the indicated time. g Normalized number of RDH molecules detected at the indicated times. The presence of Ca²⁺ results in the binding of RNase H1 to the RDH substrates without RNA degradation. Each data point is the average of three experiments. Error bars represent SD. Source data are provided as a Source Data file.