Comprehensive quantification of RNA base stacking and its determinants

The National Institute of General Medical Sciences, National Institutes of Health, awarded The Research Foundation For The State University Of New York $191,000 on August 10, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to quantify RNA base-stacking energetics and their functional consequences under physiologically relevant conditions.

The recipient will conduct systematic measurements of stacking energies across all 16 RNA base-stack combinations using a high-throughput single-molecule force-clamp assay. Work encompasses three primary aims: quantifying stacking energies for each base-stack pair, determining how stacking is modulated by temperature, ionic composition, and molecular crowding, and evaluating the functional consequences of stacking in RNA ligation and strand displacement reactions. The research addresses a gap in mechanistic understanding of RNA structure, stability, and function by providing experimentally validated parameters for force-field refinement, enhancing predictive modeling of RNA folding, and linking stacking energetics to RNA-mediated processes including strand exchange, frameshifting, RNA condensation, and riboswitch function.

Performance occurs in New Paltz, New York. The period of performance runs from August 10, 2026, through March 31, 2030. This is a Project Grant, a category of assistance under NIGMS's basic research portfolio supporting fundamental discoveries that advance understanding of human health and disease.

Abstract

Base-stacking interactions are fundamental determinants of RNA structure, stability, and function, yet their energetics remain poorly quantified. Most existing estimates are indirect, derived from duplex melting, limited dinucleotide measurements, or extrapolated from DNA, and no systematic studies exist examining the influence of key physiological factors such as molecular crowding, ionic composition, or temperature. This GAP in knowledge limits the accuracy of computational models and constrains mechanistic understanding of biologically important processes including strand exchange, frameshifting, RNA condensation, and riboswitch function. This project will systematically test the hypothesis that RNA base-stacking strength is sequence-dependent and strongly modulated by cellular-like environmental conditions, and that these interactions directly influence RNA-mediated reactions. Using a high-throughput single-molecule force-clamp assay, the study will: (aim 1) quantify stacking energies across all 16 RNA base-stacks, (aim 2) determine how stacking is modulated by temperature, ionic composition, and crowding, and (aim 3) evaluate the functional consequences of stacking in RNA ligation and strand displacement reactions. These measurements will provide experimentally validated parameters for force-field refinement, enhance predictive modeling of RNA folding, and link stacking energetics to RNA- mediated processes. This research is significant because quantitative understanding of RNA stacking under physiologically relevant conditions will enable more accurate RNA structure prediction and the rational design of RNA-based nanostructures for applications including therapeutics and provide mechanistic insights into RNA function that have broad relevance to human health.