In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.

In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.

NMR of RNA - Structure and interactions.

RNA was shown to have a more substantial role in the regulation of diverse cellular processes than anticipated until recently. Answers to questions what is the structure of specific RNAs, how structure changes to accommodate different functional roles, and how RNA senses other biomolecules and changes its fold upon interaction create a complete representation of RNA involved in cellular processes. Nuclear magnetic resonance (NMR) spectroscopy encompasses a collection of methods and approaches that offer insight into several structural aspects of RNAs. We review the most recent advances in the field of viral, long non-coding, regulatory, and four-stranded RNAs, with an emphasis on the detection of dynamic sub-states and in view of chemical modifications that expand RNA's function.