Research ArticleBIOPHYSICS

Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs

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Science Advances  07 Oct 2020:
Vol. 6, no. 41, eabc6572
DOI: 10.1126/sciadv.abc6572
  • Fig. 1 RNA structures and NMR methods described in this study.

    (A) Model RNA systems: HIV-2 TAR (30 nt, 10 kDA) and hHBV ε (61 nt, 20 kDa). Residues highlighted in green are labeled with 19F-13C–5-fluorouridine (5FU) shown in the box. Green circle, 19F; brown circle, 13C; blue circle, 2H. (B) Theoretical 19F,13C spectrum showing the four observable magnetization components of the 19F-13C spin pair as well as the decoupled resonance that has the average chemical shift and linewidths of all four components.

  • Fig. 2 Theoretical R2 values for the TROSY components for 19F-13C of 5-fluorouracil (13CF) or 1H-13C of uracil (13CH).

    (A) Theoretical curves showing the expected R2 values for the TROSY component of 13CF (cyan) and 13CH (magenta) as a function of magnetic field strength (relative to 1H Larmor frequency) for τc = 6 ns (dashed line), 25 ns (solid line), and 100 ns (dotted line) at 25°C. (B) Theoretical R2 values taken at the commercially available magnetic field strength closest to the maximum TROSY effect (13CH = 950 MHz; 13CF = 600 MHz) for τc = 6, 25, and 100 ns at 25°C.

  • Fig. 3 TROSY spectra of RNA systems.

    (A) 19F-13C TROSY of 5FU HIV-2 TAR. (B) 1H-13C TROSY of WT HIV-2 TAR. (C) 19F-13C TROSY of 5FU hHBV ε. (D) 1H-13C TROSY of WT hHBV ε. The assignments of 5FU and WT TAR-2 are indicated, as well as the arbitrary peak numbers for 5FU and WT hHBV ε. The same window size was used in all four spectra to aid in comparison. Gray dashed boxes indicate signals from helical, GU, and nonhelical regions. For (D), the black box indicates a zoom-in view of poorly resolved signals.

  • Fig. 4 Measured 13C linewidths for 5FU and WT HIV-2 TAR and hHBV ε.

    Quantification of TROSY (black) and anti-TROSY (gray) (A) 13CF and (B) 13CH linewidths for HIV-2 TAR. Note that U40 was not observed in the anti-TROSY spectrum of WT HIV-2 TAR (B). In addition, the anti-TROSY component of U38 in (B) was 97 Hz and truncated to fit in the plot. Quantification of TROSY (black) and anti-TROSY (gray) (C) 13CF and (D) 13CH linewidths for hHBV ε. Note that peaks 1 through 11 in WT hHBV ε were not observed in the anti-TROSY spectrum (D). The average ± SD in Hz is shown for the TROSY and anti-TROSY components in each plot. Peak numbers and assignments are given in Fig. 3.

  • Fig. 5 Measured 19F and 1H linewidths for 5FU and WT HIV-2 TAR and hHBV ε.

    Quantification of TROSY (black) and anti-TROSY (gray) (A) 19FC and (B) 1HC linewidths for HIV-2 TAR. Quantification of TROSY (black) and anti-TROSY (gray) (C) 19FC and (D) 1HC linewidths for hHBV ε. The average ± SD in Hz is shown for the TROSY and anti-TROSY components in each plot. Peak numbers and assignments are given in Fig. 3.

  • Fig. 6 Small-molecule binding to 5FU hHBV ε.

    (A) Overlay of 19F-13C-TROSY spectra for hHBV ε without (black) and with small molecule (SM, magenta). (B) Zoom-in of nonhelical residues showing chemical shift perturbations (CSPs) upon addition of SM. (C) Quantification of the CSPs upon addition of SM. The average (Ave) CSP is shown as a dashed line.

Supplementary Materials

  • Supplementary Materials

    Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs

    Owen B. Becette, Guanghui Zong, Bin Chen, Kehinde M. Taiwo, David A. Case, T. Kwaku Dayie

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