Fluorescence resonance energy transfer as a rich source of orientational information in nucleic acid structure

Fluorescence is the most sensitive spectroscopic method used in biological systems, and is widely used to study the structure and dynamics of large biological molecules. Fluorescence resonance energy transfer (FRET) is extensively employed to measure or compare distances in such macromolecules, especially nucleic acids (DNA and RNA). Fluorescence is highly sensitive so that it can be studied at the level of a single molecule and it can be performed inside cells.

FRET results from a coupling between two fluorescent groups attached at known positions in the molecule of interest. This is through space, not requiring physical contact. The efficiency of this process depends upon the distance between the groups, and can be arranged to be in the length range that is optimal for studies of biological molecules. In studies of single molecules this can be used to analyze distances between selected segments of a molecule, and how this changes as a function of time in dynamic processes. Such information is particularly powerful in the study of DNA and RNA structure. However, a well-known complication is that the magnitude of FRET depends on both distance and orientation of the fluorescent groups. If the latter is not properly treated the distance information obtained can be very misleading. This has severely limited obtaining reliable quantitative data from FRET in the past. On the other hand, if the orientation of the fluorescent groups is understood, this can generate reliable distance estimates together with valuable angular information.

Recent work in this laboratory has demonstrated that when certain fluorescent groups (called cyanines) are used in FRET measurements in nucleic acids, they stack onto the ends of double helices where they are oriented in a defined manner. We have used this property to show that we can measure angular information from FRET data, and consequently more reliable distance information. This puts FRET measurements on a reliable quantitative basis and we now wish to develop this as a method to study the structures of DNA and RNA in solution. This should have a major impact in the structural biology and dynamic properties of nucleic acids.

The expected outcome of this work will be a methodology for the use of FRET as a structural tool for nucleic acids. This should have a major impact in the structural biology of nucleic acids, and the study of structure and dynamics in single molecules and inside cells. The immediate users are likely to be academic researchers who are studying nucleic acid structure, folding, dynamics and interactions with small molecules and proteins. Thereafter this could become equally useful to researchers in commercial laboratories. FRET-based assays are widely used in many applications, and a greater understanding of the basis of this is likely to prove beneficial. In the course of these studies we hope to perfect a robust approach for the study of the global architecture of folded nucleic acids, their dynamics and their interactions with ligands. We shall make our protocols and computer programs available to other users, principally by downloading from the internet.