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

Abstract

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 κ(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.

Structure and Dynamics of RNA Repeat Expansions That Cause Huntington’s Disease and Myotonic Dystrophy Type 1

“…The average helical rise is 2.7 Å, and the helical twist is 30.6°, slightly less compressed than average for A-form RNA (2.8 Å and −32°). (96, 97) In the ensemble of the 20 lowest-free energy structures of r(3×CAG), the conformationally stable AA mismatches adopt cis Watson–Crick/Watson–Crick pairs(98) flanked by canonical GC pairs (Figure 4). All nucleotides in the structure adopt anti χ torsion angles, which is consistent with the presence of H6/H8–H1′ sequential NOE connectivity through all residues, without large H6/H8–H1′ NOEs that are found in the syn orientation.…”

Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes

“…Förster resonance energy transfer (FRET) is a versatile tool for nucleic acid structural analyses since its efficiency sensitively depends on relative distance and orientation between a donor and an acceptor ( 3 – 13 ). In most FRET-based studies of DNA, only distance dependence has been analyzed since dye orientations are difficult to control, although several groups have succeeded in observing orientation dependence by using DNA scaffolds ( 14 – 18 ). We previously reported a novel FRET system in which donor and acceptor are incorporated into DNA through d -threoninol linkers ( 19 ).…”

Crystal Structures of Cyanine Fluorophores Stacked onto the End of Double-Stranded RNA

“…For dsDNA NMR spectroscopy has provided this conformational information, but there are no similar data for RNA. However, some of the most interesting problems that could be addressed with this methodology are probably in RNA ( 15 , 16 ), and so we want to learn how the cyanine fluorophores interact with RNA helices, and whether this differs from the interaction with dsDNA.…”