The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

General: The elucidation of multifarious molecular structures, conformational changes, thermo-dynamic 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.

Nucleic acid structures: We have developed methods for analyzing the three-dimensional structures of complex nucleic acid structures. Examples are: multi-arm DNA/RNA junctions with two, three and four (Holliday junctions) helical arms, with and without bulge nucleotides at the junction between the two helices. Fluorescence resonance energy transfer (FRET) is used for mapping the three-dimensional structures of biological macromolecules. Conformational changes are followed by employing steady-state and time-resolved fluorescence techniques. Thermodynamic conditions and molecular context features influence which structures are preferred by macromolecules. The spectroscopic properties of the chromophores depend on the context of their molecular environments, and we carry out detailed spectroscopic analyses to provide crucial data for identifying specific characteristics of the biological molecules. 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.

Macromolecular interactions: 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, and binding to proteins and lipid membrane components, are active areas of research. 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.

Pressure studies: Fluorescence spectroscopy coupled with high pressure (hydrostatic pressures up to several thousand atmospheres) reveals unique aspects of nucleosomal folding and chromosomal protein-nucleic acid complexes. Pressure perturbation over a wide range of pressures (<1 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.

Transcription: The elongation, pausing and termination phases of transcription by E. coli RNA polymerase is being investigated by several approaches. The progression of the polymerase 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, and the pause-site residence time can be controlled reversibly by hydrostatic pressure.

Imaging: We are developing fluorescence lifetime-resolved microscopy (FLIM) and endoscopy (FLIE – esp. for medical applications; e.g. tumor diagnosis). We apply 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. These techniques introduce new opportunities for quantifying and improving the discrimination of images of fluorescence molecules in biological systems. FLIM is useful for determining distributions of interacting macromolecules by FRET and concentrations of solution components (e.g. Ca++ ions), and for improving detection and discrimination in biological tissue. Our group is an integral part of the Laboratory of Fluorescence Dynamics, and all the advanced fluorescence and imaging techniques are available.