Understanding and Manipulation of Individual RNA Complexes using Single Molecule Microscopy

Angela M. Bardo, Peter Yim, Lori S. Goldner, Optical Technology Division

Eric S. DeJong, John P. Marino, Center for Advanced Research in Biotechnology



Over the last decade single molecule confocal microscopy has developed into a powerful analytical tool for the study of both biological and inorganic systems. The primary advantage of this method is the ability to eliminate ensemble averaging of chemical data thereby providing more detailed information on system dynamics and heterogeneity than is obtainable through bulk analysis. Here we are interested in using single molecule techniques to elucidate the binding and dynamics of individual RNA complexes. As a corollary to this research, we are interested in how manipulation and immobilization of these molecules can be optimized to aid our studies and further enhance nanoscale technology.

For this research we have employed a variety of spectroscopic techniques including fluorescence resonance energy transfer (FRET) and polarization modulation fluorescence. For FRET experiments, the samples are prepared by immobilizing the donor labeled RNA on a glass substrate. An acceptor labeled RNA is then allowed to hybridize to the immobilized RNA. These individual complexes are monitored and the fluorescence from both dyes is collected in two separate channels. RNA duplexes with known donor-acceptor distances are currently under investigation to determine experimental constants and to explore the extent to which heterogeneity in the FRET signal is not related to the FRET distance but rather to photophysics or surface interactions of the individual molecules. Our goal is to use this information to perform in-depth analysis of the binding kinetics of individual RNA antisense complexes.

We are also studying methods by which the individual RNA complexes can be tethered to a surface, allowing as much free diffusion as possible, while confining the molecule to the focal volume. Here, we use single molecule polarization modulation microscopy to elucidate the diffusional properties of individual dye labeled RNA complexes tethered to a glass surface. The individual RNA are tethered to the surface using biotin-streptavidin binding or succinimidyl ester chemistry favored by the biotechnology industry. The molecules are imaged using a sample scanning confocal microscopy and the fluorescence is monitored while modulating the excitation polarization. Under these conditions, dye molecules stuck to the surface will exhibit 100% fluorescence modulation at the excitation modulation frequency. Molecules free to rotate will exhibit no modulation, and molecules with motion confined by their environment will exhibit fluorescence modulation determined by the degree of steric hinderence. Samples were also studied with PEG spacers between the RNA glass surface.

Finally, in collaboration with other groups at NIST, we are developing ways to control the preparation and manipulation of these materials for use with nanofluidic devices.