Fig. 1: RNA clustering can occur without extensive sequence complementarity or significant RNA structural melting in vitro.

a Schematic and predicted structures of shu-top and shu-bottom RNAs. shu-top and shu-bottom RNAs contain split-broccoli sequences top (blue) and bottom (orange), and their intermolecular base pairing is recognized by DFHBI-1T (green or gray star) (see Supplementary Data S1 for sequences). The predicted structures were drawn by FORNA⁴⁸. b In vitro dimerization of shu-top and shu-bottom RNAs. Top panel: RNA; bottom panel: DFHBI-1T signal. The ladder was run on a different gel. c Schematics of RNA clustering of shu-top and shu-bottom RNAs that is independent (i) or dependent (ii) of base pairing generated by extensive sequence complementarity or significant structural melting. The two models are distinguished by the absence (gray circle (i)) or presence (green circle (ii)) of DFHBI-1T fluorescence. d, e In vitro RNA clusters (magenta) formed of only shu-top or shu-bottom RNAs (negative controls), co-folded (positive control) or separately folded shu-top or shu-bottom RNAs (e) in the presence of DFHBI-1T (green). The DFHBI-1T images in (d, e) were normalized using the parameters of the co-folded DFHBI-1T image (see also Supplementary Fig. S1d for images normalized to the DFHBI-1T signal of shu-top). Initial RNA concentrations: 3.2 μM (negative controls), 1.6 μM for each RNA in co-folding and separate folding. Scale bars: 7.5 μm. f Normalized intensity of DFHBI-1T by RNA intensities (Supplementary Fig. S1a–c) from outside (gray) or inside (green) in RNA clusters per condition. n = 30 RNA clusters for each condition. Data: Mean ± SEM. n.s.: not significant. p-values (unpaired t test; two-tailed): 0.30 (shu-top), 0.47 (shu-bottom), 0.30 (co-folding) and 0.089 (separate folding). See also Supplementary Fig. S1 and Supplementary Data S1. Source data are provided as a Source Data file.