Structural Biochemistry/Mechanical Unfolding of RNA
Mechanical unfolding of RNA has become the preferred method by which to study the RNA folding problem, due to its lack of need for high temperatures or denaturants. The invention of laser tweezers has now made this technique even more valuable, in that it allows force to be applied to single molecules of RNA, and a clear view of their unfolding and refolding. To discover the structural transitions of RNA, measured force is applied and the end-to-end distance of the strand is measured; this also allows for the calculation of mechanical work done. Also developed was the technique FRET, or fluorescence resonance energy transfer, which is used to make clear RNA folding conformational change kinetics (Li, Vieregg, Tinoco Jr., 78).
When studying metal ion bonding, mechanical unfolding is the preferred method of study for several reasons. The use of optical tweezers allows RNA to be studied in physiological conditions, and the force applied by the tweezers only affects non-covalent interactions. Because of this, thermodynamic calculations and interpretations remain simple; the force applied affects only the RNA's structure, and not the activity of water molecules and ions (85). Therefore, scientists do not need to consider colligative properties in their experiments. Furthermore, RNA conformation can be manipulated so that scientists can measure the formation or the break of interaction in RNA. Overall, the development of this method has resulted in easier, more accurate studying of RNA structure.
Li, Pan T.X., Jeffrey Vieregg, and Ignacio Tinoco. "How RNA Unfolds and Refolds." Annual Review of Biochemistry 77.1 (2008): 77-100. Print.