RNA circularization preserves dynamics and enables in-cell relaxation NMR.

RNA function is governed by transient structural rearrangements that are sensitive to the cellular environment. While NMR spectroscopy provides unique access to RNA dynamics at atomic resolution, such measurements are typically restricted to in vitro conditions due to rapid degradation of unmodified RNA by intracellular RNases. Here, we develop a modular and efficient enzymatic pipeline for circularizing hairpins to enhance stability and enable in-cell NMR studies of RNA dynamics. The circular RNA exhibits a lifetime of >24 h in lysate, comparable to fully 2'-O-methylated RNA, without requiring chemical modifications. Circularization also preserves the secondary structure of four biologically and structurally diverse RNAs as well as maintaining native excited-state dynamics for two of the constructs. Interestingly, circularization of RNAs whose dynamics involve interhelical bending reveals a transfer of dynamics across RNA helices, reminiscent of allosteric cooperativity. The enhanced intracellular stability enables the first reproducible in-cell measurements of imino proton relaxation rates on chemically unmodified RNA. Together, this work establishes RNA circularization as a broadly applicable strategy for quantitative in-cell NMR studies of RNA structure and dynamics, and uncovers a possible mechanism for long-range cooperativity in RNA.