Extended Data Fig. 5: Analysis of the oligomeric state of NbaSPARDA by size-exclusion chromatography and multi-angle light scattering.
a, Analysis of the WT SPARDA complex by SEC-MALS. The absolute molar weight of the NbaAgo/DREN-APAZ heterodimer is indicated. The Mw/Mn polydispersity index was 1.006, suggesting particles with the same Mw across the peak. b, Analysis of the MID mutant of NbaSPARDA by SEC-MALS, with the absolute molecular weight indicated. The Mw/Mn polydispersity index was 1.000. c, Size-exclusion chromatography of the purified WT NbaSPARDA complex in the absence of nucleic acids (top), in the presence of 20 nt gRNA (middle) and in the presence of 20 nt gRNA and 20 nt tDNA (bottom). For each sample, OD280 and OD260 absorbance profiles are shown. OD500 profiles are also shown for samples containing gRNA (which was labeled with a FAM residue at its 3’-end, see Supplementary Table 1). In each panel, positions of monomeric (apparent Mw of 83 or 96 kDa) and dimeric (apparent Mw of ~150 kDa) SPARDA peaks are indicated. Note that gRNA binding slightly increases the apparent size of SPARDA, as suggested by an increase of the apparent Mw from 83 to 96 kDa and hydrodynamic radius from 3.88 to 4.12 nm, but does not involve any distinct oligomeric state transition. d, Analysis of the apparent hydrodynamic radii and masses of monomeric (heterodimers of NbaAgo and DREN-APAZ) and dimeric (dimers of heterodimers) SPARDA complexes, based on calibration with standard albumin samples. e, Analysis of nucleic acids from various fractions after size-exclusion chromatography of the SPARDA sample pre-incubated with gRNA (20 nt) and tDNA (20 nt) (from the bottom profile in panel C). In each case, the results of a single experiment are shown. Based on the OD260/OD280 ratio (panel c) and the electrophoretic analysis of gRNA and tDNA in each fraction (panel e) it can be concluded that dimers of the heterodimeric SPARDA complex contain only gRNA, while monomeric heterodimers contain both gRNA and tDNA. Thus, the dimeric peak of SPARDA observed in the presence of gRNA (middle profile in panel c) or gRNA and tDNA (bottom profile in panel c) likely corresponds to gRNA-bound SPARDA. The presence of a minor dimeric fraction in WT SPARDA (top profile in panel c and bottom profile in panel d) likely results from binding of gRNAs co-purified with SPARDA, while the main peak corresponds to gRNA-free monomeric SPARDA.
