Label-Free, High-Time Resolution, Single-Molecule Studies of RNA Folding and Dynamics

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RNA is a class of biosynthetic molecules that plays central roles in gene expression, the process of using the genetic information stored in DNA to make functional proteins. How RNAs function in gene expression is dependent on their specific and complex three-dimensional shapes or structures and on their ability to undergo precisely timed and executed structural changes (their dynamic rearrangements). With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Ruben Gonzalez and Dr. Colin Nuckolls from Columbia University to study how RNAs fold and/or undergo structural rearrangements and how the dynamics of a particular RNA structural ‘switch’ controls the expression of a human gene. Drs. Gonzalez’s and Nuckolls’ research team uses carbon nanotube-based field-effect transistors (smFETs) to follow the detailed dynamics of individual RNA molecules in three-dimensional space and across time. Notably, the smFET method allows the study of RNA dynamics without the need to chemically modify the RNA or apply invasive external forces. A deeper understanding of how RNA-based structural switches function to control gene expression impacts the discovery and development of therapeutics that act on important biomolecular targets and/or functions. In addition, the project increases participation by women and underrepresented minorities in STEM by leveraging existing outreach programs in New York City. Kits are developed to demonstrate nucleic acid structures and single-molecule biophysics in K-12 community outreach, while education modules are developed to teach integrated concepts of chemistry, biology, and computer science at local high schools. Using smFETs, Drs. Gonzalez’s and Nuckolls’ research team investigate the kinetic mechanisms with which RNA stem-loops, a fundamental class of RNA structures, fold and undergo functionally important, dynamic structural rearrangements at unparalleled molecular, spatial, and temporal resolutions. The resulting model is used to determine the mechanism through which the naturally occurring, post-transcriptional methylation of the N6 position of an adenosine in the stem of a RNA stem-loop found within a human precursor messenger RNA (pre-mRNA) regulates the dynamics of this RNA stem-loop as a means for controlling the splicing of the pre-mRNA. Specifically, the proposed studies characterize the folding and dynamics of this stem-loop and tests the hypothesis that the post-transcriptional, N6 methylation of the adenosine alters the dynamics of the stem-loop (i.e., throws the ‘switch’ on) so as to unmask the binding site of an RNA-binding protein that directs the differential splicing of the pre-mRNA. The results of these studies provide a general model for the function of RNA stem-loop based conformational switches and a detailed understanding of the mechanism through which a post-transcriptional modification of a stem-loop controls the alternative splicing of a pre-mRNA.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.