Research Interests
The elucidation of multifarious molecular structures, conformational changes, thermodynamic stabilities and functions of nucleic acids and protein/nucleic acid complexes is a major research topic in our laboratory. We employ a variety of techniques, such as fluorescence spectroscopy, rapid kinetic methods and physical perturbations in order to probe the intra- and intermolecular interactions of these macromolecular structures, and to understand the physical basis of their biological functions. Modern biotechnology makes it possible to synthesize tailored molecules that represent many aspects of the natural bio-molecular systems, and permits us to relate our physical measurements to specific selected characteristics of biological systems.
We have developed methods for analyzing the three-dimensional structures of complex nucleic acid structures. Examples are: multi-arm DNA/RNA junctions with two helical arms (with bulges of different sizes at the junction between the two helices), three arms (with and without bulges at the crossover point of the junction), four-way junctions (Holliday genetic recombination junctions) and ribozymes (e.g. hammerhead ribozyme). Fluorescence resonance energy transfer (FRET) is a major experimental tool for mapping the three-dimensional structures of biological macromolecules under diverse biologically relevant conditions. Conformational changes, brought about by temperature or changing solution conditions, can be followed by employing various fluorescence techniques, such as steady-state and time-resolved intensity and anisotropy measurements. There are many thermodynamic and solution conditions, as well as molecular context features, that influence which particular structures are preferred by macromolecules under different conditions. Our spectroscopic methods are ideal for investigating miscellaneous molecular properties. We carry out detailed spectroscopic analyses of the fluorophores that we attach covalently to macromolecules. The properties of the chromophores depend on the context of their molecular environments, and these spectroscopic investigations are necessary to establish reliable prerequisites for proper interpretations of the fluorescence/structural experiments. This quantitative spectroscopic information also provides crucial data for correlating the fluorescence measurements with identifiable characteristics of the biological molecules, such as the specific nucleic acid sequence. Many interesting, fundamental characteristics of these complex nucleic acid structures have been revealed by our fluorescence studies. We are presently investigating characteristic structural, dynamic and thermodynamic features of vital elements that make up essential components of key RNA molecules, such as ribozymes, different components of ribosomal RNA.
Chromosomal protein-nucleic acid complexes are also being studied. Fluorescence spectroscopy coupled with high pressure (hydrostatic pressures up to several thousand atmospheres) have revealed unique aspects of nucleosomal folding. Pressure perturbation over a wide range of pressures (from fractions of an atmosphere to several thousand atmospheres) constitutes one of our staple experimental methods. Applications range from studies of macromolecular conformational changes to studies of biological cells and inactivation of pernicious viruses, such as SIV and HIV. Pressure perturbation of biological systems is a very valuable and unique method and is an exciting area of research with wide applications.
The spectroscopic, thermodynamic and kinetic characteristics of intercalating and groove binding drugs, such as Hoechst, DAPI, ethidium, acridines, and other small molecules, to DNA and RNA molecules is an active area of our research. Similar investigations with proteins and lipid membrane components are also of interest. Of special relevance for our studies is the use of rapid kinetic methods, such as stopped-flow and the relaxation techniques (temperature-jump and pressure jump). Such kinetic studies make it possible to disperse the separate "normal mode" components of a reaction mechanism on the time axis, revealing details of the individual steps of a reaction mechanism. These studies not only give us insights into the interactions of small molecules with macromolecular structures but also supply us with valuable information pertaining to the macromolecules themselves, such as their molecular dynamics and the occupancy of different populations of their conformational states.
The elongation, pausing and termination phases of transcription by E. coli RNA polymerase is being investigated by several approaches. For instance, the progression of the polymerase enzyme along a template can be followed by fluorescence, the rate of the reaction can be influenced by dye- and drug-binding to the template and RNA product, and the catalytic reaction steps can be tuned with pressure, temperature and small interacting molecular components. These experiments have shown directly that RNAP enzyme molecules exist in solution as individual entities with dissimilar properties, and that the rate of nucleotide incorporation can be controlled significantly by simply controlling physical solution characteristics, such as high pressure or the binding of small molecules to the template. The synthesis of RNA can be halted completely at less than 1000 atmospheres pressure, and the reaction returns fully reversibly to the canonical rates when the pressure is released (the RNAP remains fully processive). The residence at pause sites is differentially affected by pressure. Many aspects of the transcription reaction become amenable to study using such general physical, and yet specifically acting, perturbations and selective reaction conditions. Very sensitive methods of fluorescence detection make even single molecule experiments possible.
We also have a very active program involving the development of various types of advanced instrumentation. For instance, one major project involves the development of fluorescence lifetime-resolved microscopy (FLIM) and the application of this new imaging method to a variety of biological problems, such as the detection of tumor cells and their discrimination from healthy cells by identifying specific fluorescence lifetimes. This project also involves the extension of the imaging methods to fiber-optic endoscopes with the concomitant medical applications (e.g. tumor diagnosis). These techniques introduce new opportunities for quantifying and improving the discrimination of images of fluorescence molecules in biological systems. FLIM has been shown to be very useful for determining distributions of interacting macromolecules by FRET, determining concentrations of solution components (e.g. Ca++ ions) and improving detection in biological tissue. All the advanced fluorescence and imaging techniques in the Laboratory of Fluorescence Dynamics are available for our studies, as our group is an integral part of this research resource.
Selected references: