The rapidly increasing characterization of RNA tertiary structures has revealed their pervasiveness and active roles in human diseases. Small molecule-mediated modulation of RNA tertiary structures constitutes an attractive avenue for the development of tools for both therapeutically targeting and/or uncovering the pathways associated with these RNA motifs. This potential has been highlighted by preliminary targeting of the triple helix present at the 3'-end of the non-coding RNA MALAT1, a transcript implicated in several human diseases. This triplex has been reported to decrease the transcript susceptibility to degradation and, ultimately, promote its cellular accumulation. While small molecules have been shown to bind and impact the stability of the MALAT1 triple helix, the small molecule properties that lead to these structural modulations are not well understood. To elucidate these properties, we designed a focused library utilizing the diminazene scaffold, which is under-explored but precedented for nucleic acid binding, to target the MALAT1 triple helix. We then employed multiple assays to holistically assess what parameters, if any, could predict small molecule affinity and effect on triplex stability. We designed and/or optimized competition, calorimetry, and thermal shift assays as well as an enzymatic degradation assay, the latter of which led to the discovery of bidirectional modulators of triple helix stability within the scaffold-centric library. Determination of quantitative structure activity relationships (QSAR) afforded predictive models for both affinity- and stability-based assays. This work establishes a suite of powerful orthogonal biophysical tools for the evaluation of small molecule:RNA triplex interactions that generate predictive models and will allow small molecule interrogation of the growing body of disease-associated RNA triple helices.
- car
- Ggplot2
- Glmnet
For more information, please refer to our paper: Coming soon!
We are grateful to current and past Hargrove lab members, especially to G. Padroni and S. Wicks, for their feedback, input, and support ultimately shaping the scope and framework of this study. We are grateful to the Tolbert lab for their generous donation of the T7 polymerase used for in vitro transcription. We thank the head of the NMR core B. Bobay, and of the MS core P. Silinski. We would also like to thank J. Brown for kindly sharing illustrator files to make 2D-renderings of the MALAT1 triple helix. TOC graphic was made with BioRender.
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