Research in the Qin group focuses on understanding the structure/function relationship on nucleic acids. We are utilizing a unique biophysical tool, called site-directed spin labeling (SDSL), for studying nucleic acids. At USC, we have developed new SDSL methodologies driven by specific questions related to DNA and RNA, and these expanded SDSL tools have provided information where other methods have failed. The SDSL method, in combination of other biochemical and biophysical approaches, are being utilized to investigate specific DNA and RNA molecules that function in fundamental biological processes. These studies are aimed at revealing detailed information regarding the conformations of the molecules, as well as unveiling the links between structure, dynamics and function. The information will aid in understanding basic biological processes, deciphering causes of human diseases, and developing novel nano-scale devices. Currently, major areas of research focus include:
Site-directed spin labeling studies of nucleic acids.
In SDSL, a stable nitroxide radical attached to a specific site is utilized to provide local structural and dynamic information via electron paramagnetic resonance (EPR) spectroscopy. SDSL has been established as a tool for investigating solution structure and dynamics of proteins, especially for those systems that are difficult to study by X-ray crystallography and NMR spectroscopy. SDSL studies of nucleic acids, although having shown great potentials, lag behind the more mature protein SDSL applications. We are among the first group of researchers to use SDSL to study folded RNAs, and we are continuing SDSL developments as driven by specific biological problems.
Our current work focuses primarily on a family of nitroxides, called the R5-series, which are covalently attached to the backbone of a target nucleotide within an arbitrary DNA or RNA sequence. We have reported R5 labeling methods that are simple, efficient, and cost-effective both in terms of time and expense. With these advantages, we can “scan” the nitroxide through a given nucleic acid sequence. This is advantageous for obtaining comprehensive information regarding the target macromolecule.
We have reported a R5-based SDSL tool-kit for unrestricted mapping global structures of nucleic acids. It allows one to use state-of-the-art pulse EPR techniques to measure nanometer distances (20 – 50 Å) between a pair of R5’s attached in DNA and RNA molecules. The measure distances are then correlated to the parent nucleic acid structure using an efficient, web-accessible program, called NASNOX (linked via our group site http://pzqin.usc.edu/pzqhome/). We are currently applying this tool-kit to probe global structures of nucleic acids and protein/nucleic acid complexes.
Another area of investigation focuses on probing nucleic acids via monitoring the dynamics (i.e., rotational motion) of a singly-attached nitroxide (e.g., R5). Using this approach, we are probing nanosecond dynamics in large folded RNA molecules (e.g., a 400-nucleotide group I intron), as well as investigating local structural and dynamic features in DNAs.
Structure and function of the packaging RNA, an essential component in the strongest known biological motor.
The pRNA is an essential component of the phi29 bacteriophage DNA packaging motor, which condenses the linear DNA genome inside a capsid utilizing forces that are 2 ~ 8 fold higher than other motors such as myosin and RNA polymerase. Multiple copies of pRNA form a ring-shape complex that is essential in the motor ATPase activity. Information on pRNA structure and function is limited. This has hindered the studies of the phi29 motor, which is believed to share the same mechanism as other double-stranded DNA viruses, including human pathogens such as herpes simplex viruses.
At USC we have carried out biochemical analyses of a pRNA closed dimer, which is the simplest functionally relevant pRNA ring-shaped complex. The results have revealed a modular architecture in the pRNA, and led to a “rigid body motion” model, where ATP dependent movements within a pRNA monomer sequentially activate each monomer and drive DNA packaging. To test the “rigid body motion” model, structure, assembly, and motion of pRNA are being investigated with a combination of biochemical and biophysical methods. SDSL methods, particularly the R5-tool kit, are the major component in these investigations.
As a component of a nano-motor evolved by nature, pRNA provides a framework for constructing novel, artificial nano-machines. Our work on pRNA structure and interaction will aid in these developments.
Non-canonical nucleic acid structures probed via SDSL.
A particular interest is non-B DNA conformations, which associate either with key gene expression and/or regulation events or with human diseases. Studies on non-B conformations in solution are generally hampered by the large size of the target molecules and the co-existence of B and non-B conformations. SDSL is capable of probing heterogeneous high-molecular weight systems, and is being used to test specific hypotheses regarding non-B conformations in a human sequence. The studies use a combination of methods, including EPR spectral measurement, EPR spectral simulations, molecular dynamics simulations, and mutagenesis, to establish rules for correlating the nitroxide dynamics to the local nucleic acid environment. Using these rules, observed nitroxide spectra are analyzed to assess the conformations of the molecule as well as to monitor conformational changes.