24] Application of circular dichroism to study RNA folding transitions

Abstract

Circular dichroism (CD) spectroscopy has been widely used to study secondary structure formation in both proteins and nucleic acids. In this chapter, the application of CD is used to study RNA folding transitions. Modern commercially available CD spectrometers are capable of simultaneous CD and absorbance measurements. The amplitude of the applied photomultiplier voltage (high tension) directly relates to absorbance of the sample and is recorded along with the CD signal. This enables to measure transitions by both probes without any additional measurements. The chapter also discusses the use of CD in combination with Mg2+ or urea titrations to study the secondary and tertiary structural transitions in RNAs. Two-domain ribozyme P RNA have been from Bacillus subtilis folds with two distinct structural transitions, an unfolded to intermediate, U-to-I, transition and an intermediate to native transition. The U-to-I transition occurs at micromolar Mg2+ concentrations and is readily monitored by changes in absorbance (A260) and CD at 260 nm. These signals are primarily sensitive to the formation of helical structure. Tertiary transition occurs in millimolar Mg2+ range with a Hill coefficient of about 4. The formation of the tertiary structure of RNA is the result of structural changes involving the ribosephosphate backbone and non-Watson–Crick base pairing of nucleotides.