LAUSR

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 how we used smFRET to show that single spliceosomes - responsible for the accurate removal of all intervening sequences (introns) in pre-m(essenger) RNAs - are working as biased Brownian ratchet machines. Second, we have developed Single Molecule Cluster Analysis (SiMCAn) based on a large smFRET dataset to further dissect the complex conformational dynamics of the pre-mRNA as it is spliced. 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 cancer. Microinjected, singly-fluorophore labeled, functional miRNAs were tracked at super-resolution within individual diffusing particles. Observed mobility and mRNA dependent assembly changes suggest the existence of two kinetically distinct assembly processes. We are currently feeding these data into a single molecule systems biology pipeline to bring into focus the unifying molecular mechanism of such a ubiquitous gene regulatory pathway.