Extended Data Fig. 7: Evidence of multimer formation of GOLLD, ROOL, and OLE in biologically relevant concentrations.

(a) Agilent Bioanalyzer traces demonstrate the purity of the samples. The second peak for OLE is a common artifact of poor denaturation of sample in Bioanalyzer traces. The pure monomeric reading in mass photometry, (b), shows that this peak is likely not a covalently linked dimer. (b) Mass of GOLLD, ROOL, and OLE complexes as obtained from mass photometry at 50 nM, 50 nM, and 12.5 nM respectively. The data is a histogram of particle count density, normalized per sample, where dark is many counts, white is none. Total particle counts are shown above the graph. (c) Hydrodynamic radius of GOLLD and ROOL complexes as derived from dynamic light scattering at 110 nM and 140 nM respectively. The data are plotted as relative population density, normalized by density per sample, with dark representing highly populated radius values. The temperature of the sample was raised from 25 °C to 75 °C and dynamic light scattering traces were obtained every 10 °C, showing complex melting into monomers at 65 °C and aggregation at high temperatures. (d) Representative ratiometric image for all mass photometry data (1 frame from a 60 s collection at 331 Hz). (e) Mass photometry data of OLE in different buffer conditions demonstrates OLE can dimerize at low RNA concentration, low magnesium concentration, and in the absence of magnesium with sufficient monovalent cations. (f) The mass photometric data is summarized by counting the amount of hits in the monomer, dimer, and high stoichiometry peaks. The absolute ratio of monomer:dimer is accurate as assessed in (g-h). (g) Mass photometry traces of mixtures of ROOL and GOLLD, ratiometric image examples can be found in. (h) Summary of the mixture results, with the known complex ratio plotted against the ratio reported by mass photometry. There is agreement, but with slight bias towards higher counts for the smaller species, ROOL, opposite of the previously observed trend⁷⁵.