Figure 3.

Chemical exchange in CCR5 revealed by ¹³C Carr–Purcell–Meiboom–Gill relaxation dispersion (CPMG RD) experiments. a) 2D ¹H–¹³C HMQC spectrum of a ligated CCR5 RNA sample labeled only with site-specific (8,1′-¹³C)-adenosines within the donor fragment. This was recorded in the absence of miR-1224, at 298 K, at 600 MHz proton Larmor frequency, with T_relax=0 ms. The expected eight anomeric H-C cross-peaks are well-resolved. b) ¹³C CPMG RD profiles of the ligated CCR5 RNA at 150 MHz ¹³C Larmor frequency. Red dots represent experimental data and black dots represent repeated experiments. Solid lines are best-RD-curve fits of the CPMG profiles. Residues A75, A76, A88, A90, and A95 show non-flat dispersion profiles whereas A85 and A96 show flat dispersion profiles. Note that the A79 resonance was too broad to adequately fit. c) ¹³C CPMG RD profiles of A90 ribose anomeric carbon atom of the ligated CCR5 RNA at 150 (red dots) and 200 MHz (blue dots) ¹³C Larmor frequency. Black circles represent repeated experiments and solid lines are the best fits of the CPMG profiles. d) NMR titration of the ligated CCR5-A90 RNA with miR-1224. The 2D HMQC spectrum in magenta recorded in the absence of miR-1224 exhibits a single cross-peak corresponding to ribose C1′ of the residue A90. Upon addition of miR-1224, the ribose A90-C1′ chemical shift changed dramatically from 90.3 ppm to 88.3 ppm (in cyan). The large chemical shift change (up to 2.0 ppm) indicates that the interaction with miR-1224 alters the local chemical environment of the A90 residues. In the presence of an equimolar amount of miR-1224, the CCR5 RNA is not completely converted to its bound form, with ca. 9% remaining in the free conformation. This is consistent with the EMSA result (Figure S6). However, adding 20% extra amount of miR-1224 converts it into the completely bound form (data not shown).