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. 2022 Apr 18;298(6):101952. doi: 10.1016/j.jbc.2022.101952

Figure 3.

Figure 3

tDR-GlyGCC binding affinity to HDL by microscale thermophoresis.A, setup of microscale thermophoresis (MST). Fluorescently labeled (AF647) target (e.g., single stranded sRNA oligos) were heat denatured (90 °C for 2 min) and combined (1:1) with serial ligand dilutions (e.g., DGUC-HDL). Oligo-ligand mix were then loaded in glass capillaries, and MST was performed where an infrared laser heats the complex to create a small temperature gradient. sRNAs with HDL will demonstrate slower movement compared to free sRNA molecules. The movement of the fluorescent sRNA is then tracked. B, example of a binding curve depicting difference between unbound sRNAs and bound sRNA molecules thermophoretic movement, where Fnorm is the normalized fluorescence against the ligand concentration, ΔFnorm is the baseline corrected normalized fluorescence. Data were fitted to the MST (Kd) model. Mean MST analysis from triplicate assays are presented, Kd are mean ± SD. C, MST analysis of interactions between tDR-GlyGCC-30 and a scrambled antisense (AS) oligonucleotide (AS-Scr) or a complementary antisense to tDR-GlyGCC-30 (AS-tDR-GlyGCC-30). D, MST binding curves of tDR-GlyGCC-30 and DGUC-HDL. E, MST analysis of interactions between DGUC-HDL and tDR-GlyGCC-30, tDR-GlyCCC-30, or tDR-LysCTT-30. F, binding curves of DGUC-HDL to single-stranded (ssRNA) or double-stranded (dsRNA) tDR-GlyGCC-30 without (nondenatured) or with heat (90 °C 2 min) denaturing (denatured). G, schematic of tDR-GlyGCC fragments synthesized used in (H). H, MST analysis of interactions between DGUC HDL and tDR-GlyGCC fragments in (G). Electrophoretic mobility shift assay (EMSA) with fluorescently (AF546) labeled sRNA and DGUC-HDL. tDR-GlyGCC (AF546) was incubated with titrations of HDL (1:1) and crosslinked (@254 nm) for 15 min. Samples were loaded onto a 4–16% NativePAGE gel with 0.5× TBE running buffer. I, Coomassie of nondenaturing gel. Stokes radius (Rs) of proteins standards shown. J, fluorescent (AF546) migration of tDR-GlyGCC-30 on gel with free oligo and oligo complexed to HDL titrations is shown. Schematics created with BioRender.com. DGUC, density-gradient ultracentrifugation; HDL, high-density lipoprotein; tDR, tRNA-derived sRNA; sRNAs, small RNAs.