NMR Characterization of an Oligonucleotide Model of the MiR-21 Pre-Element

Abstract

We have used NMR spectroscopy to characterize an oligonucleotide stem loop structure based on the pre-element of an oncogenic microRNA, miR-21. This predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. It is also a critical feature recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA. The secondary structure of the native sequence is poorly defined by NMR due to rapid exchange of imino protons with solvent; however, replacement of two adjacent putative G•U base pairs with G•C base pairs retains the conformation of the hairpin observed by chemical probing and stabilizes it sufficiently to observe most of the imino proton resonances of the molecule. The observed resonances are consistent with the predicted secondary structure. In addition, a peak due to a loop uridine suggests an interaction between it and a bulged uridine in the stem. Assignment of non-exchangeable proton resonances and characterization of NOEs and coupling constants allows inference of the following features of the structure: extrahelicity of a bulged adenosine, deviation from A-form geometry in a base-paired stem, and consecutive stacking of the adenosines in the 5′ side of the loop, the guanosine of the closing base pair, and a cross-strand adenosine. Modeling of the structure by restrained molecular dynamics suggests a basis for the interaction between the loop uridine, the bulged uridine in the stem, and an A•U base pair in the stem.

A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21 Precursor and Suppresses Dicer Processing

“…In 15 N- 1 H HSQC spectra recorded at 5C, we also observed four weak and broad peaks with chemical shift values corresponding to tandem U31:G44/G32:U43 pairs that breathe and open frequently, leading to rapid exchange with solvent and peak broadening (figure 1D). Altogether, the NMR data are consistent with the relaxed structure suggested by mutational analysis, NMR, SHAPE, in-line probing and nuclease digestion 7, 23, 35, 40, 41, 44 .…”

“…To date, high resolution structural information on pre-miRNA hairpins has been limited to computational models or mutated structures 40–43 . Surprisingly, only one wild type pre-miRNA hairpin loop structure (miR-20b) is deposited in the PDB 43 .…”

Visualizing a protonated RNA state that modulates microRNA-21 maturation

“…1c). This observation is consistent with previous NMR studies on miR-21 precursor 33,34 , where mutations of the pre-element were made to quench the structural flexibility into a single conformational state 33 . When we carried out an NMR 13 C-1 H HSQC experiment that probes nonsolvent-exchangeable signals, surprisingly only 20 out of a total of 29 expected NMR resonances from preE-miR-21 were observed at room temperature ( Fig.…”

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

“…Increased avidity of 3 is observed to an RNA that contains both the A and U bulges (K d = 22 ± 8.3 nM) (Figure S2). Despite the dynamics of RNA structure, both the A and U bulge regions in the miR-21 hairpin precursor have been observed by NMR (Chirayil, et al, 2014). Additional binding analyses using microscale thermophoresis (MST) (Moon, et al, 2018; Seidel, et al, 2013), indicated the binding affinity of 3 to a miR-21 hairpin containing both the A and U bulge (miR-21 Hairpin; Figure S2D) and a miR-21 hairpin containing only the A bulge (miR-21 A Bulge; Figure S2E) as 500 and 300 nM, respectively.…”