Elucidating microRNA-34a organisation within Human Argonaute-2 by DNP MAS NMR

Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, deregulated in various cancers, in hAgo2, effector protein in RISC, using guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced solid-state NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and then bound to SIRT1 mRNA compared to the free hairpin or the free duplex formed with mRNA. Nucleotide-specific information of the of C2- and C3-endo sugar puckering can be obtained from the C8 - C1 correlation with varying distributions, revealing a trapping of different confirmations upon freezing. C3-endo puckering was predominantly observed for the seed, while C2-endo for the central region and a mixture of both elsewhere. These observations provide insights into the molecular dynamic basis of miR-based mRNA regulation, while also providing a proof-of-concept that experiments under cryogenic conditions, e.g. at 90K, can trap and with that reveal frozen dynamic states, using methods such as (DNP-enhanced) solid-state NMR or Cryo-EM