Small Molecule Modulation of RNA Tertiary Structures and RNA-Protein Interactions

Abstract

The recently characterized therapeutic promise of noncoding RNAs has led to renewed vigor in the development of targeting strategies to manipulate their biological functions. While there have been strides in using primary sequence-based approaches to base-pair to specific regions of interest, small molecules offer unique promise in targeting RNA due to their favorable pharmacokinetics and synthetic accessibility. RNA-targeting small molecules have been used successfully in targeting more complex RNA secondary and tertiary structures, thereby altering function. However, due to the inherent flexibility and dynamics of RNA, there is a general lack of mechanistic understanding of how small molecules manipulate RNA conformational space to affect these functions. Among these biological functions is the ability to bind trans-acting factors, chief among them RNA-binding proteins, which can also modulate and shift the equilibrium of RNA conformations through their binding interactions. Therefore, developing a holistic understanding of how small molecules impact RNA structural space and thereby influence protein interactions can inform the next generation of RNA-targeting small molecule therapeutics.To this effect, we assessed an RNA-targeting focused library for interactions with an RNA triple helix at the 3'-end of the cancer-associated long noncoding RNA MALAT1. We evaluated these interactions using affinity-based assays as well as applying an in vitro RT-qPCR assay to measure small molecule impacts to structural stability. In an effort to explore the cheminformatic properties driving these interactions, we developed quantitative structure-activity relationship models for small molecule binding and stability shifts. We further employed these models to predict and select novel molecules to test the model’s predictive power for functional outputs. This study demonstrates the feasibility of using these models to inform synthetic selection with predictive function, as well as providing high affinity probes for future cellular targeting of MALAT1. To evaluate RNA-binding proteins and how they interact with different RNA structures, we applied stability-based mass spectrometry proteomics to evaluate changes in protein stability upon RNA addition. Using the aforementioned noncoding RNA triple helix, a viral RNA stem-loop, and single stranded RNA sequence from the untranslated region of a coding mRNA, we used thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX) to interrogate RNA-protein interactions on the proteomic scale. We found that these methods, which require heat and denaturant to shift the protein folding equilibrium, can shift the equilibrium of the RNA conformational space, leading to the identification of RNA-binding protein hits that did not arise in more common pull-down methods. These tools provide novel avenues to interrogate RNA-binding proteins in a greater RNA dynamic context and could also be applied to the study of RNA-small molecule targeting to evaluate changes in protein stability through small molecule-induced RNA conformational shifts. We holistically evaluated small molecule-induced conformational change and influence on protein binding through the scope of RNA G-quadruplexes. We investigated the targetability and modulation of RNA G-quadruplexes using our RNA-targeting small molecule focused library, as well as measured how these molecules impact G-quadruplex-protein interactions. By identifying small molecules which could either stabilize or unfold a model RNA G-quadruplex, we then applied the SPROX workflow to identify how proteins shifted in stability in the presence of the G-quadruplex and when small molecules were added. With the application of mass spectrometry, we compiled a comprehensive list of proteins which change in stability upon small molecule addition, suggesting impacts to these RNA-protein interactions in a small molecule dependent manner. This work is the first to highlight this dynamic relationship between small molecule, protein, and RNA G-quadruplex conformational space on the proteomic scale. The work discussed herein provided insights into small molecule properties which lead to differential RNA-small molecule recognition and structural modulation. Additionally, this work highlights the use of stability-based mass spectrometry tools to assay dynamic RNA-protein interactions. Taken together, these advances will boost future RNA-targeting efforts with tools to investigate how small molecules both modulate RNA conformational dynamics and further manipulate RNA-protein interactions.