Extended Data Fig. 3: DGR RNA Binding and cPRT Base Pairing.

a-d. Electrophoretic mobility shift assay (EMSA) of (a) DGR RNA, (b) DGR RNA Δ^avd368-379, (c) DGR RNA Δ^Sp96-140, and (d) non-DGR RNA with varying concentrations of bRT-Avd (as indicated at top of each panel). RNA was resolved by 4% native PAGE. Arrowheads indicate position of shifted bands. To the side of each gel is shown LC/MS-MS analysis of shifted EMSA bands. Lower quantities of RNA at higher bRT-Avd concentrations in the non-DGR RNA sample likely indicate non-specific aggregation of bRT-Avd with RNA. For gel source data, see Supplementary Fig. 1b–e. Representative gel from two independent replicates is shown. e. Schematic of (1, WT) 12 potential bps in the avd-Sp duplex; (2) substitutions in Sp that disrupt the 12 bps; (3) complementary substitutions in avd that restore the 12 bps; (4) substitutions in Sp that disrupt six Watson-Crick bps; and (5) complementary substitutions in avd that restore the six Watson-Crick bps. Solid bars are Watson-Crick base pairs, and open circles wobble base pairs. f. Top, cDNA synthesis by bRT-Avd with (1) wild-type or (2-5) mutant DGR RNA. The lane numbers correspond to the numbering in panel e. Products were treated with RNase and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane M corresponds to radiolabeled, single-stranded DNA molecular mass markers, denoted by nucleotide (nt) length. Arrowheads indicate the positions of ~90- and ~120-nt cDNAs. A representative gel from three independent replicates is shown. Bottom, input RNA for cDNA synthesis resolved by 6% denaturing PAGE. Lane M corresponds to DNA molecular mass markers, denoted by nt length. DGR RNA runs at a position higher than the 300 nt marker, likely due to retention of secondary structure in the RNA. The numbers correspond to the numbering of sequences depicted in panel e. A representative gel from three independent replicates is shown. For gel source data, see Supplementary Fig. 1f.