Unveiling Functionally Critical, Ephemeral RNA (un)folding States with Magnetic Tape Head Tweezers

RNA, or ribonucleic acid, is the molecular cousin of DNA, the genetic blueprint of the cell, and bears a seemingly minor difference in chemical composition.

This difference, however small, together with the absence of a second, complementary strand, enables single RNA molecules to fold into structures that can be as intricate as those of proteins.

This folding lays the foundation for a multitude of cellular RNA functions, particularly controlling and executing gene expression, including of viruses and bacteria.

The current project will investigate the kinetics and thermodynamics with which a foundational set of RNA structures, ranging from a hairpin found in the human immunodeficiency virus (HIV) to a pseudoknot and a long-range docking architecture found in two bacterial RNAs, fold and undergo functionally important structural rearrangements from single base pairs to the entire RNA molecule.

Bridging the associated broad time and length scales will be achieved using a novel magnetic tape head pulling approach to interrogate individual surface tethered RNA molecules, with the goal of conquering a long-standing challenge in understanding biologically important RNA structure-dynamics-function relationships― that of the coupling of short- with long-range fluctuations.

Specifically, the hypothesis will be tested that the formation of individual base pairs guides the formation of large helical elements, which in turn govern the accessible topology as the RNA folds.

It is anticipated that the results will provide the basis for general models of RNA folding while also inspiring a diverse group of high school and undergraduate students getting involved in this research to pursue a STEM degree.<br/><br/><br/>RNA molecules are unique among biopolymers in that they couple inheritable sequence information with the ability to fold into complex three-dimensional structures with important biological functions in gene regulation.

After decades of study, the precise links of RNA sequence with folding and function are only starting to emerge, in part due to the challenging range of scales exhibited by RNA folding – fast, sub-millisecond base pair fluctuations give rise to minute-slow, large-scale conformational rearrangements.

The current project introduces a novel experimental platform for RNA studies, capable of interrogating this entire time and length regime relevant to RNA (un)folding.

Specifically, preliminary data demonstrate the use of a magnetic tape head force spectrometer to pull for hours at microsecond time resolution on single superparamagnetic-bead tethered RNA molecules, using a wide, physiologically relevant force range of 0 to 50 pN.

This project will focus on three long-studied gene regulatory RNAs of increasing structural complexity: the HIV TAR hairpin, the small preQ1 riboswitch pseudoknot, and the four-way junction Mn2+ riboswitch.

Combined with computational modeling, this set of targets is anticipated to help reveal the contributions of sequence and ligand binding to RNA (un)folding in unprecedented detail.

Complementarily, a multi-pronged approach will be pursued to involve traditionally underrepresented high school and undergraduate students in research, aiming for an impact in the nearby city of Detroit.

This project thus aims to leverage technology to present educational research activities that engage the broader public in a scientific topic – RNA – cast into the spotlight by the COVID-19 pandemic.<br/><br/>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.