In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.
Dipanwita BanerjeeHisae Tateishi-KarimataMaria ToplishekTatsuya OhyamaSaptarshi GhoshShuntaro TakahashiMarko TrajkovskiJanez PlavecNaoki SugimotoPublished in: Journal of the American Chemical Society (2023)
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.
Keyphrases
- nucleic acid
- single molecule
- living cells
- energy transfer
- gene expression
- induced apoptosis
- fluorescent probe
- circulating tumor
- magnetic resonance
- cell cycle arrest
- genome editing
- crispr cas
- cell free
- atomic force microscopy
- small molecule
- oxidative stress
- magnetic resonance imaging
- stem cells
- endoplasmic reticulum stress
- single cell
- signaling pathway
- quantum dots
- cell death
- risk assessment
- computed tomography
- bone marrow
- high resolution
- transcription factor
- heat shock