"Single Molecules Come Into Focus: Understanding RNA-Driven Regulation From First Principles"
This event is part of the Biophysics Seminars. 12:30PM.
Nature and Nanotechnology likewise employ nanoscale machines that self-assemble into structures of complex architecture and functionality. Fluorescence microscopy offers a non-invasive tool to probe and ultimately dissect and 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. First, I will describe a tool we developed termed Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) that detects the conformational changes of single riboswitch containing bacterial mRNAs upon sensing the metabolite preQ11. Second, I will illustrate how we demonstrated that ligand binding modulates cross-coupling between riboswitch folding and transcriptional pausing by the bacterial RNA polymerase. Third, I will describe a method for the intracellular single molecule, high-resolution localization and counting (iSHiRLoC) of microRNAs (miRNAs), a large group of gene silencers with profound roles in our body, from stem cell development to cancer2-6. Single microinjected, singly-fluorophore labeled, functional miRNAs were tracked at super-resolution within individual diffusing particles, revealing their sub-cellular trafficking. Finally, I will show how Single-Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) can precisely count single microRNA molecules in human blood serum as a new detection paradigm of clinical relevance.