Measuring nanometer distances in the packaging RNA using Double Electron-Electron Resonance.
EMSL Project ID
21494
Abstract
We request instrument time on the EMSL pulse electron paramagnetic resonance (EPR) system in order to measure nanometer distances in nucleic acids using the Double Electron-Electron Resonance (DEER) method. My laboratory studies nucleic acids structure and interaction utilizing the site-directed spin labeling technique, in which local structural and dynamic information is obtained via EPR spectroscopy of a stable nitroxide radical attached site-specifically to a bio-molecule. Using the DEER method, we have measured distances between 2 to 4 nm in DNA and RNA with a nitroxide probe that can be attached, in an efficient and cost-effective manner, to arbitrary sequences in nucleic acids. With NSF and NIH funding, the scope of our experiments has drastically expanded, and locally available pulse EPR instrumentation time does not meet our demand. We request a total of 600 hours for the EMSL pulse EPR instrument time from October 2006 to April, which amounts to an average of one week per month. The requested instrument time will be used to measure distances in the packaging RNA (pRNA), which is an essential component in the strongest known biological motor - the phi29 bacteriophage DNA packaging motor. The experiments will provide quantitative information on the global structure of the pRNA, which has been difficult to study using X-ray crystallography or NMR spectroscopy. They represent the first application of pulse EPR to map the conformation of a large RNA molecule with no prior high-resolution structure, and will establish a new method for structural probing on nucleic acids. The results will advance our understanding of pRNA structure and function in the packaging motor. This might aid in designing new nano-motors, thus supporting the needs of DOE and the nation. The proposed work fits very well with the EMSL science theme on "Biological Interactions and Interfaces", and is expected to produce publications in high impact journals such as Nature Structural and Molecular Biology and Proceedings of the National Academy of Sciences.
Project Details
Project type
Capability Research
Start Date
2006-10-24
End Date
2008-09-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Grant GPG, N Boyd, D Herschlag, and PZ Qin. 2009. "Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling." Journal of the American Chemical Society 131(9):3136-3137. doi:10.1021/ja808217s
Popova A, T Kalai, K Hideg, and PZ Qin. 2009. "Site-Specific DNA Structural and Dynamic Features Revealed by Nucleotide-Independent Nitroxide Probes." Biochemistry 48(36):8540-8550. doi:10.1021/bi900860w
Zhang X, CS Tung, G Sowa, MM Hatmal, IS Haworth, and PZ Qin. 2012. "Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling." Journal of the American Chemical Society 134(5):2644-2652. doi:10.1021/ja2093647