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

Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove (13)C-(13)C dipolar couplings that complicate (13)C relaxation analyses. While these phenomena are well documented for sites with adjacent (13)C 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) (13)C-(13)C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-(13)C/(15)N]-ATP or selectively [2-(13)C]-ATP labeled RNAs. Our simulations predict non-negligible (13)C-(13)C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R(1)) relaxation rates in [U-(13)C/(15)N]-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 R(1) measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-(13)C/(15)N]-ATP or [2-(13)C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range (13)C-(13)C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R(1) rates in terms of motional models for large RNAs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10858-021-00368-8.