Consolidated 3-Fold Isotopic Lens for Probing RNAs

Undesired scalar and dipolar couplings are two major interactions that complicate structural and dynamic studies in solution nuclear magnetic resonance (NMR) spectroscopy. Recent developments in site-specific isotopic labeling technologies have gone a long way toward alleviating these problems. While some nuclei have intrinsic properties that make them suitable for specific NMR experiments, these same properties render them inefficient in other experiments. Site-specific isotopic labeling facilitates the controlled incorporation of isotopes to enable facile analysis of RNAs. Here, we describe the synthesis and incorporation of [1′-¹³C, 2-¹⁹F, 7-¹⁵N] adenosine 5′-triphosphate into the 9 kDa Escherichia coli rRNA, thus expanding the applications of previously synthesized [2-¹³C, 7-¹⁵N]-adenosine 5′-triphosphate, with the added benefit of ¹⁹F incorporation. We utilized these ¹³C and ¹⁵N probes to characterize the structural dynamic features within this RNA, and ¹⁹F was used to monitor binding interactions. Finally, we leveraged the chemical shielding anisotropy-dominated relaxation of ¹⁵N7-adenosine for straightforward analysis of R₁ and R_1ρ rates.