Efficient Detection of Hydrogen Bonds in Dynamic Regions of RNA by Sensitivity-Optimized NMR Pulse Sequences

摘要

In recent years, RNA has emerged as a central player in the regulation of gene expression in all kingdoms of life, ranging from sRNA in bacteria to miRNAs and long noncoding RNAs in eukaryotes.While the sequences and biochemical properties of these RNAs are typically well established, their three-dimensional structures and structure-function relationship are often poorly characterized. Hydrogen bonds (H-bonds) define the secondary and tertiary structures of nucleic acids (Figure la). Thermodynamics-based structure prediction and covariational methods are useful theoretical approaches but require experimental validation. Biochemical structure probing methods can provide such information, even for very large RNAs, but the analysis is complicated by alternate structures or by the presence of conformational dynamics. NMR-spectroscopy allows the identification of base pairs in nucleic acids based on the detection of H-bonds involving imino protons, which are shielded from exchange with bulk water. NOE-correlations of imino protons establish base pairing in helical regions of nucleic acids, but only provide indirect evidence for H-bonds.