Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA

Abstract

Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that complicate 13C relaxation analyses. While these phenomena are well documented for sites with adjacent 13C nuclei (e.g. ribose C1′), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å) 13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible 13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1 measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range 13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1 rates in terms of motional models for large RNAs.

NMR of RNA - Structure and interactions

“…13 C CEST experiments were further pushed to detect faster motions using high-power radio-frequency fields [61]. These studies were complemented with a detailed assessment of the effect of carbonecarbon coupling on relaxation parameters measured in samples with different labeling schemes [62] and with quantification of the effect of 13 C dipolar coupling on relaxation rates, which become non-negligible, especially at the higher magnetic field and large molecular weights of RNAs [63]. The high-pressure NMR was also shown to be useful for the detection of low-abundance excited states of RNA [64].…”

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

“…The RNA motions reported by R 1 , R 2 , and hNOE are easily probed by 13 C − ,− and 15 N ,, nuclei. 15 N sites are present in the four nucleobases at the following sites: adenosine (Ade)-H2-N1, Ade-H2-N3, Ade-H8-N7, and Ade-H8-N9, guanosine (Gua)-H1-N1, Gua-H8-N7, and Gua-H8-N9, uridine (Uri)-H3-N3, and Uri-H6-N1, and cytidine (Cyt)-H6-N1 (Figures and ).…”

“…Therefore, 13 C spins do not approximate an isolated two-spin system. In uniformly labeled samples, these extensive dipolar couplings complicate 13 C R 1 rate measurements and analysis ,,,,,,− in biopolymers of large size (τ C > 7 ns). Given this fact, our group has developed pulse schemes (based on the isolated 1 H– 15 N backbone amide spin pair in proteins) to leverage the isolated 1 H– 13 C spin pairs afforded by our atom-specifically labeled RNA samples (Figure ).…”

“…(B) Simulated R 1 rates and R 1 difference (defined as above) for the nuclei highlighted in panel A. R 1 simulations were carried out for 800 MHz field and R 1 difference simulations were run at multiple magnetic fields. All simulations were carried out at various τ C values, and additional details can be found in the original works. , …”

“…Experimental measurements with our customized pulse sequence (for selectively labeled RNA) (Figure 5) and those of others 186 (for uniformly labeled RNA), corroborated our simulations, suggesting that these discrepancies in R 1 cannot be wholly ignored, even for fairly isolated Ade-C2 and Pur-C8 sites. 188,189 Taken together, the contribution of 13 C− 13 C dipolar interactions needs to be explicitly taken into consideration in data analysis of uniformly labeled RNA. Spin relaxation measurements on uniformly labeled RNA from Al-Hashimi and co-workers 186 demonstrate that this is not an insurmountable hurdle.…”