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

Abstract

A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (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 (<Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (k_PS) and backward (k_SP) 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

Keywords:

• m3U force field
• RNA hairpin
• ACSL region of tRNAs
• NMR kinetics
• RNA structure

Acknowledgements

Synthesis, instrumental characterization, and HPLC analysis of the 17-mer m3U7-RNA hairpin were presented as a preliminary RNA study in the Ph.D. thesis of Cenk A. Andac (School of Pharmacy, Institute of Health Sciences at Gazi University-Turkiye, thesis ID: 260803; https://tez.yok.gov.tr/UlusalTezMerkezi/giris.jsp) and at the 3^rd International Conference and Exhibition on Advances in Chromatography & HPLC Techniques, July 13-14, 2017 Berlin, Germany. NMR kinetics and MD data presented in this manuscript merely represent post-thesis studies. We wish to express our gratitude to Dr. Stephen A. Scaringe (the former founder of Dharmacon Inc., Lafayette, CO, USA) for providing us with a batch of m3U7-RNA for cross-checking. The idea of the presence of m3U7 in the ACL region of hypothetical tRNAs to study primordial RNA replication belongs to Prof. Ulfert Hornemann (School of Pharmacy, University of Wisconsin, Madison WI USA), who served as a former Ph.D. advisor of the main author (Cenk A. Andac) and as the primary investigator in this RNA study. This work is dedicated to the memory of Prof. Ulfert Hornemann, who died of cancer in 2010. We would like to thank Turkish Science e-Infrastructure (TRUBA) for providing us with additional computational resources. We greatly acknowledge Prof. David Case (Rutgers University, Piscataway, NJ, USA) and AMBER administrators for granting us AMBER suite of programs v18 and v20.

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