CCR5 RNA Pseudoknots: Residue and Site-Specific Labeling correlate Internal Motions with microRNA Binding
| Autor: | Christoph Kreutz, Andrew P. Longhini, Bin Chen, Jonathan D. Dinman, Felix Nußbaumer, T. Kwaku Dayie |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Models Molecular Receptors CCR5 Chemistry Chemical shift Organic Chemistry Relaxation (NMR) RNA General Chemistry Non-coding RNA Catalysis Homonuclear molecule Article 03 medical and health sciences MicroRNAs 030104 developmental biology Biophysics Molecule Nucleotide Pseudoknot Nuclear Magnetic Resonance Biomolecular Solid-Phase Synthesis Techniques |
| Zdroj: | Chemistry |
| Popis: | Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution-state NMR are difficult for large RNA molecules (> 70 nt; nt = nucleotides) owing to severe spectral overlap, homonuclear (13)C scalar couplings, and line broadening. Herein, a strategic combination of solid-phase synthesis, site-specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position-specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA-1224 complex indicated that A90-C1′ of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR-labeling strategy to probe functional RNA structural dynamics. |
| Databáze: | OpenAIRE |
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