研究领域

Analytical Chemistry Bioanalytical Chemistry Bioinorganic Chemistry Biophysical Chemistry Chemical Biology Energy Science Nano Chemistry Optics and Imaging Organic Chemistry Physical Chemistry RNA BioChemistry Sensor Science Surface Chemistry Sustainable Chemistry Ultrafast Dynamics

At the interface of Chemistry and Biology, a revolution has recently taken place that has uncovered a plethora of small non-coding (nc)RNAs in our bodies, which outnumber protein-coding genes by several-fold, dominate the expression patterns of all genes in all cells, and have inspired entirely new therapeutic intervention approaches. Our group's goal is to understand the structure-function relationships in these ncRNAs using single molecule tools and then utilize them for biomedical, bioanalytical and nanotechnological applications. The ncRNAs we study range from small RNA catalysts, such as the hammerhead, hairpin and hepatitis delta virus ribozymes with potential use in human gene therapy and relevance to human disease, to large RNA-protein complexes, such as RNA interference machinery involved in gene regulation and virus suppression. In particular, we employ fluorescence techniques to study in real-time the kinetic mechanisms of these ncRNAs, in bulk solution, in live cells, and at the single-molecule level. Applications include the identification and optimization of ribozymes for gene therapy and as novel biosensors, as well as the characterization of antiviral and antibiotic drugs that target pathogenic RNA function. Our research by its very nature is highly interdisciplinary, engaging students with a diverse background and providing a broad education. The molecules we study are extremely dynamic over time scales of microseconds to hours. To understand these dynamics we combine state-of-the-art chemical, molecular biological, and biophysical approaches. An outline of several exciting current projects is given below. 1. Developing a model system for understanding gene silencing by directly observing, using fluorescence techniques, the action of small interfering (si)RNAs and micro (mi)RNAs on pathogenic mRNAs in cell extracts and live cells. 2. Utilizing single molecule fluorescence imaging techniques to follow movement of the ribosome on a secondary structured mRNA, including riboswitch motifs that utilize an aptamer domain to recognize a specific ligand and effect downstream gene expression. 3. Utilizing super-resolution fluorescence imaging techniques in nanotechnology to follow autonomously moving engineered "molecular spiders". 4. Developing a non-biological mimic of the superb sensing and actuation efficiency and precision reached during RNA transcription. 5. Using single-molecule fluorescence techniques to observe in unprecedented detail fluctuations of single ribozyme molecules between catalytically active and inactive conformations. 6. Dissecting pre-mRNA splicing by fluorophore labeling individual RNA or protein components and following their fluorescence fluctuations during splicing in cell extracts by single molecule fluorescence microscopy.

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Bartke, R.M., Cameron, E.L., Cristie-David, A.S., Custer, T.C., Denies, M.S., Daher, M., Dhakal, S., Ghosh, S., Heinicke, L.A., Hoff, J.D., Hou, Q., Kahlscheuer, M.L., Karslake, J., Krieger, A.G., Li, J., Li, X., Lund, P.E., Vo, N.N., Park, J., Pitchiaya, S., Rai, V., Smith, D.J., Suddala, K.C., Wang, J., Widom, J.R. and Walter, N.G. (2015). Meeting report: SMART timing-principles of single molecule techniques course at the University of Michigan 2014. Biopolymers 103, p. 296-302. Full text in pdf format. Sripathi, K.N., Banas, P., Reblova, K., Sponer, J., Otyepka, M., and Walter, N.G. (2015). Wobble pairs of the HDV ribozyme play specific roles in stabilization of active site dynamics. Phys. Chem. Chem. Phys. 17, p. 5887-5900. Full text in pdf format. Nyati, S., Schinske-Sebolt, K., Pitchiaya, S., Chekhovskiy, K., Chator, A., Chaudhry, N., Dosch, J., Van Dort, M.E., Varambally, S., Kumar-Sinha, C., Nyati, M.K., Ray, D., Walter, N.G., Yu, H., Ross, B.D. and Rehemtulla, A. (2015). The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-b signaling. Sci. Signal. 8, ra1, p. 1-11. Full text in pdf format. Supplementary Information in pdf format. Mlynsky, V., Walter, N.G., Sponer, J., Otyepka, M. and Banas, P. (2015). The role of an active site Mg2+ in HDV ribozyme self-cleavage: insights from QM/MM calculations. Phys.Chem.Chem.Phys. 17, p. 670-679. Full text in pdf format. Rinaldi, A.J., Suddala, K.C. and Walter, N.G. (2015). Native purification and labeling of RNA for single molecule fluorescence studies. Methods Mol. Biol. 1240, p. 63-95. Full text in pdf format. Pitchiaya, S., Androsavich, J.R. and Walter, N.G. (2014). Super-accuracy and super-resolution fluorescence microscopy based on single molecule detection. In Imaging Life: Chapter 9 , pp. 228-259 (G. C. Howard, W. E. Brown, and M. Auer, eds.), Oxford University Press, New York. Full text in pdf format. Suddala, K.C. and Walter, N.G. (2014). Riboswitch structure and dynamics by smFRET microscopy. Methods Enzymol. 549, p. 343-373. Full text in pdf format. Widom, J.R., Dhakal, S., Heinicke, L.A. and Walter, N.G. (2014). Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update. Arch. Toxicol. 88, p. 1965-1985. Full text in pdf format. Fu, J., Yang, Y.R., Johnson-Buck, A., Liu, M., Liu, Y., Walter, N.G., Woodbury, N.W. and Yan, H. (2014). Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 9, p. 531-536. Full text in pdf format. Supplementary Information in pdf format. Access the recommendation on F1000Prime Johnson-Buck, A., Jiang, S., Yan, H. and Walter, N.G. (2014). DNA-cholesterol barges as programmable membrane-exploring agents. ACS Nano 8, p. 5641-5649. Full text in pdf format. Supplementary Information in pdf format. Sripathi, K.N., Tay, W.W., Banas, P., Otyepka, M., Sponer, J. and Walter, N.G. (2014). Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape. RNA 20, p. 1112-1128. Full text in pdf format. Johnson-Buck, A. and Walter, N.G. (2014). Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy. Methods 67, p. 177-184. Full text in pdf format. Harris, D.A., Todd, G.C. and Walter, N.G. (2014). Terbium(III) footprinting as a probe of RNA structure and metal binding sites. In Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition, Part II, pp. 255-268 (R.K. Hartmann, A. Bindereif, A. Schon, and E. Westhof, eds.), Wiley-VCH Verlag, Weinheim. Full text in pdf format. Walter, N.G. and Bustamante, C.J. (2014). Introduction to single molecule imaging and mechanics: seeing and touching molecules one at a time. Chem. Rev. 114, p. 3069-3071. Full text in pdf format. Pitchiaya, S., Heinicke, L.A., Custer, T.C. and Walter, N.G. (2014). Single molecule fluorescence approaches shed light on intracellular RNAs. Chem. Rev. 114, p. 3224-3265. Full text in pdf format. Access the recommendation on F1000Prime Grima, R., Walter, N.G. and Schnell, S. (2014). Single-molecule enzymology a la Michaelis-Menten. FEBS J. 281, p. 518-530. Full text in pdf format. Suddala, K.C., Rinaldi, A.J., Feng, J., Mustoe, A.M., Eichhorn, C.D., Liberman, J.A., Wedekind, J.E., Al-Hashimi, H.M., Brooks III, C.L. and Walter, N.G. (2013). Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure. Nucleic Acids Res. 41, p. 10462-10475. Full text in pdf format. Supplementary Information in pdf format. Krishnan, R., Blanco, M.R., Kahlscheuer, M.L., Abelson, J., Guthrie, C. and Walter, N.G. (2013). Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat. Struct. Mol. Biol. 20, p. 1450-1459. Full text in pdf format. Supplementary Information in pdf format. Pitchiaya, S., Krishnan, V., Custer, T.C. and Walter, N.G. (2013). Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting. Methods 63, p. 188-199. Full text in pdf format. Walter, N.G. (2013). The bright future of (non-coding) RNAs: methods light the way. Methods 63, p. 93-94. Full text in pdf format.