Sub-Millisecond RNA Collapse Observed in a Microfluidic Mixer

Abstract

Measuring the fast collapse of RNA molecules can elucidate the first steps of RNA folding. The P5abc sub-domain of the Tetrahymena ribozyme is an example of an RNA molecule that folds rapidly following the addition of Mg2+ ions. We studied changes in truncated-P5abc (tP5abc) RNA as reported by Forster Resonance Energy Transfer (FRET) in a microfluidic mixing device using confocal microscopy. With sub-millisecond time resolution, we measured the ion species and valence dependent collapse timescales of tP5abc. In this presentation, we discuss differences between collapse times of tP5abc and those of a mutant which does not fully fold in the presence of magnesium ions. We also discuss experimental issues that arise when using an internally labeled fluorophore to report changes in RNA conformation. To account for changes in the FRET efficiency we consider results of fluorescence anisotropy measurements, as well as fluorescence correlation spectroscopy measurements.