Ligand-induced conformational changes observed in single RNA molecules

Abstract

We present the first demonstration that f luorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg 2؉ ions were studied by changes in single-molecule f luorescence. The transition from an open to a folded configuration was monitored by the change of f luorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg 2؉ concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg 2؉ concentration with 20-ms time resolution, making it the world's smallest Mg 2؉ meter.The study of individual molecules allows one to look beyond ensemble-averaged properties. For example, one can measure the distributions of physical properties and observe the detailed time trajectories of molecular conformations. The study of single molecules has also led to the discovery that identical DNA molecules under identical conditions follow a multitude of paths as they extend in elongational flows (1). This ''molecular individualism'' was shown to be a consequence of thermal variations in the initial random polymer coil before extension and of nonequilibrium statistical mechanics (2). It is possible that complex processes such as protein folding and enzyme activity will also show a rich set of kinetic paths that can be fully characterized only at the single-molecule level. Thus, it is important to develop techniques that extend our ability to observe such molecular processes at this level of sensitivity.Fluorescence resonance energy transfer (FRET) (3, 4) provides a powerful way of observing the behavior of molecules. Donor and acceptor dyes attached to two sites of a biological molecule can be used to measure the distance between the two dyes. Donor fluorescence emission is strongly quenched in a distance-dependent manner by the acceptor, whereas the acceptor emission increases because of the energy transfer. Thus, measurement of a change in fluorescence from the two dyes can be used as an indicator of a change in the conformation of the host molecule. Because the two fluorophores are on different parts of the molecule, intramolecular motion can be measured in the mol...

Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

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Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…Early single molecule FRET studies on RNA folding kinetics revealed a wealth of information inaccessible to traditional pre-steady state kinetic methods, demonstrating the utility of these studies [117][118][119]. Since then, there has been an explosion of single molecule fluorescence studies on RNA [120].…”

Mg 2+ -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

“…A confocal scanning microscope system was used to excite and detect fluorescent-labeled single RNA junctions as described (17). Donors were directly excited with an argon laser (Coherent, Santa Clara, CA) at 514 nm, and fluorescence was collected through an oil immersion objective (Achroplan 100ϫ͞1.25 oil; Zeiss) and detected at the donor emission maximum (590 nm) and acceptor emission maximum (675 nm) by two photon counting modules (SPCM-AQ, EG & G Optoelectronics, Vaudreuil, Quebec).…”

“…When fluorescence resonance energy transfer (FRET) (14,15) was applied to single molecules (16), we previously observed conformational changes of individual RNA junctions that contained shortened helices 20, 21, and 22 (17). In the open conformation, the donor and acceptor are about 8.5 nm apart, whereas in the folded conformation they are about 5 nm apart based on the three-dimensional structure (10,11).…”