Synthesis, NMR kinetics and dynamic structure of a 17-mer heptaloop RNA hairpin carrying a 3-<i>N</i>-methyluridine nucleotide residue in the loop region

A 17-mer RNA hairpin (5'GGGAGU<b>X</b>AGCGGCUCCC3') carrying 3-<i>N</i>-methyluridine (m3U) at position <b>X</b> (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. ¹H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (∼5-6%) ACSL conformations. Exchange rate constant (k_ex) for interconversion between P and S states is 112 sec⁻¹ (&lt;Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (k<i>_PS</i>) and backward (k<i>_SP</i>) rate constants are 49.166 sec⁻¹ and 62.792 sec⁻¹, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by ¹H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs. Communicated by Ramaswamy H. Sarma