Quantifying the effects of long-range ¹³C-¹³C 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 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.