(598b) Understanding Accessibility for Targeting Nucleic Acids in Living Cells Via a Thermo-Kinetic Model of in Vivo Oligonucleotide Hybridization

RNAs continue to be pursued as major regulatory targets to control cellular regulation for therapeutic and biotechnology applications. To this end, tuning of protein expression at the mRNA level by synthetic complementary anti-sense RNAs (asRNAs) has gained significant traction. A major requirement for this approach is the design of asRNAs that can effectively bind target mRNAs inside the dynamic environment of living systems. In designing these asRNAs, a major factor that is often excluded is the structure of the target RNA. To address this limitation, we used an in vivo experimental system (in vivo RNA Structural Sensing System-iRS³) to study RNA-RNA interactions using a fluorescence reporter as a measure of RNA binding. Then, we derived a biophysical model that describes RNA binding and incorporates the structural accessibility of the target region. We used the model to investigate the importance of structural accessibility in the context of other kinetic and thermodynamic factors. This study demonstrates that thermodynamics of the interaction alone do not explain patterns of asRNA binding in living cells. Instead, our model shows that nucleotide accessibility within the binding region is a stronger indicator of the effectiveness of asRNA complementation. Collectively, our results suggest that binding of an asRNA to a highly structured target region seems to be driven predominantly by kinetic factors rather than thermodynamics. Therefore we conclude that accounting for nucleotide accessibility is key for designing asRNAs that can effectively target an RNA of interest.