Ligand-induced conformational changes observed in single RNA molecules

We present the first demonstration that fluorescence resonance energy trans fer can be used to track the motion of a single molecule undergoing conform ational changes. As a model system, the conformational changes of individua l three-helix junction RNA molecules induced by the binding of ribosomal pr otein S15 or Mg2+ ions were studied qv changes in single-molecule fluoresce nce. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Ave raged behavior of RNA molecules closely resembles that of unlabeled molecul es in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic aci ds interactions. Surprisingly, we observed an anomalously broad distributio n of RNA conformations at intermediate ion concentrations that may be attri buted to foldability differences among RNA molecules. In addition, an exper imental scheme was developed where the real-time response of single molecul es tan be followed under changing environments. As a demonstration, we repe atedly changed Mg2+ concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instant aneous Mg2+ concentration with 20-ms time resolution, making it the world's smallest Mg2+ meter.