RNA: Tools for Cell Biology

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

This year we reported the development of an RNA-based intramolecular fluorescence resonant energy transfer (FRET) reporter that can provide information not only on distance but angular orientation between two fluorophores. Because it results from dipole-dipole interaction, FRET is a function of both, distance and orientation (as well as spectral overlap) between two fluorophores. Although FRET is widely employed in studies of proximity and interaction of biomolecules, both in vitro and in vivo, usually only the distance-dependence is used. This is because most fluorophores are attached to proteins and nucleic acids through flexible linkages. Due to thermal motion, the orientation of the fluorophores relative to the biomolecules under study is largely randomized, making analysis of the relative orientation of the fluorophores very difficult. Our laboratory has structurally characterized fluorescent RNA aptamer-fluorophore complexes. We reasoned that since RNA, especially in its double-stranded form, is extremely rigid, placing two fluorescent RNA modules at appropriate locations in an RNA under study could yield FRET reporters on the structure and structural transition of the RNA of interest. We constructed a model system in which two fluoromodules whose structures we have determined are separated by variable numbers of base pairs. Consistent with structure-based modeling, the FRET intensity varied in a damped sinusoidal function of the number of base pairs intervening between the fluorophores. Appropriate deconvolution allows extraction of the orientation component. We successfully tested the approach further in characterizing the ligand dependent re-organization of a bacterial gene-regulatory RNA. Overall, this approach will be of value for characterizing large RNA and RNA-protein complex machines, using time-, distance-, and orientation-dependent FRET.