Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5' terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter ?(2) as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.