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.
Highlights
RNAs are important macromolecules that function in a wide range of cellular roles (Cech and Steitz 2014; Mortimer et al 2014; Sharp 2009)
We recently showed that purine C8 may experience non-negligible dipolar contributions to R 1 relaxation from non-adjacent coupling partners (Nam et al 2020)
We demonstrate that the removal of long-range 13C-13C dipolar coupling partners reveals discrepancies in measured adenosine C2 R1 values between uniformly and selectively labeled samples
Summary
RNAs are important macromolecules that function in a wide range of cellular roles (Cech and Steitz 2014; Mortimer et al 2014; Sharp 2009). RNAs are dynamic and can sample numerous conformations on various time scales that might be important for function (Zhao et al 2017; Marušič et al 2019). Three commonly measured dynamics parameters are the longitudinal ( R1) and transverse (R2) relaxation rates and the heteronuclear Overhauser effect (hNOE) (Marušič et al 2019; Palmer 2004; Wagner 1993). The R1 rate measures the return of the longitudinal magnetization to thermal equilibrium whereas R 2 measures the decay of transverse magnetization, and the hNOE measures the change in heteronuclear spin magnetization in response to saturating proton spins (Yamazaki et al 1994; Peng and Wagner 1992; Abragam 1961)
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