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Nils Walter, University of Michigan

Host - Ruobo Zhou (865-5610) 

"Single Molecules Come Into Focus: From Riboswitches to Molecular Diagnostics"

Abstract: At least 75% of the 3 billion base pairs of the human genome are transcribed into RNA, but the vast majority of these transcripts do not code for proteins but rather for “non-coding” RNAs (ncRNAs), many of which remain uncharacterized in terms of their structure and function¹. Currently, more than 80,000 unique ncRNAs have been identified in human cells alone, suggesting that for a long time we have underestimated the intricacies involved in human genome maintenance, processing, and regulation by neglecting this far-reaching “RNA World.”  Nature and modern nanotechnology likewise employ nanoscale RNA machines that self-assemble into structures of complex architecture and functionality.  Fluorescence microscopy offers a non-invasive tool to probe, dissect and ultimately control these nanoassemblies in real-time.  In particular, single molecule fluorescence resonance energy transfer (smFRET) allows us to measure distances at the 2-8 nm scale, whereas complementary super-resolution localization techniques based on Gaussian fitting of imaged point spread functions (PSFs) measure distances in the 10 nm and longer range^2,3.  Encapsulating the power of these recent technical advances, we have combined single-molecule and biochemical approaches to show that a central, adaptable RNA helix in the widespread manganese-sensing riboswitch functions analogous to a molecular fulcrum to integrate disparate signals for finely balanced bacterial gene expression control⁴.  We posit that many more examples of such intimate structural and kinetic coupling between RNA folding and gene expression remain to be discovered, leading to the exquisite regulatory control and kinetic proofreading enabling all life processes.  On the more applied side, we are developing tools to detect single RNA, DNA and protein molecules as potential biomarkers of disease^5,6.

References

1. Walter, N. G. (2015). Going viral: riding the RNA wave to discovery. RNA 21, 756-7. PMC-ID: 4371365.

2. Jalihal, A. P., Lund, P. E. & Walter, N. G. (2019). Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope. Cold Spring Harb Perspect Biol 11. PMC-ID: in progress.

3. Ray, S., Widom, J. R. & Walter, N. G. (2018). Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World. Chem Rev 118, 4120-4155. PMC-ID:

4. Chauvier, A. & Walter, N. G. (2024). Regulation of bacterial gene expression by non-coding RNA: It is all about time! Cell Chem Biol 31, 71-85. PMC-ID:

5. Johnson-Buck, A., Su, X., Giraldez, M. D., Zhao, M., Tewari, M. & Walter, N. G. (2015). Kinetic fingerprinting to identify and count single nucleic acids. Nat Biotechnol 33, 730-2. PMC-ID:

6. Mandal, S., Li, Z., Chatterjee, T., Khanna, K., Montoya, K., Dai, L., Petersen, C., Li, L., Tewari, M., Johnson-Buck, A. & Walter, N. G. (2021). Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers. Acc Chem Res 54, 388-402. PMC-ID: