Time-resolved mechanisms of essential biological pathways.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9912914

Exploring cleavage activity of NSP15 using single molecule PIE-FRET

Large numbers of inanimate biomolecular machines organize across space and time and undergo drastic changes in shape to carry out complex biological processes. The detailed molecular mechanisms of these tightly regulated pathways are rarely known but driven by series of highly coordinated protein-protein and protein-nucleic acid interactions. Catching these transient events in action requires fast detection and high sensitivity, which is offered by time-resolved fluorescence methods. I will discuss single molecule time-resolved fluorescence microscopy and spectroscopy as a tool to explore in real time the interactions and structural rearrangements that drive specific pathways. We are currently studying the mechanisms of ribosome assembly, SARS-CoV-2 viral RNA processing, and DNA mismatch repair. We have designed assays for simultaneous Föster resonance energy transfer and fluorescence correlation spectroscopy measurements to investigate several fundamental questions. In my talk, I will present recent findings that reveal the dynamic interactions between essential enzymes, assembly factors, and nucleic acids. We aim to completely characterize the impact of mutations on the molecular function of enzymes to understand how complex diseases develop at the molecular level and to identify new therapeutic targets. 

Revealing pathways of ribosome assembly and viral RNA processing with time-resolved fluorescence

Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics across many timescales. With TCSPC, Fluorescence correlation spectroscopy (FCS) and pulsed interleaved excitation-Förster resonance energy transfer (PIE-FRET) are collected simultaneously on diffusing molecules to extract diffusion characteristics and proximity information. This article is a guide to calibrating FCS and PIE-FRET measurements with several biological samples including liposomes, streptavidin-coated quantum dots, proteins, and nucleic acids for reliable determination of diffusion coefficients and FRET efficiency. The FRET efficiency results are also compared to surface-attached single molecules using fluorescence lifetime imaging microscopy (FLIM-FRET). Combining the methods is a powerful approach to revealing mechanistic details of biological processes and pathways.

A practical guide to time-resolved fluorescence microscopy and spectroscopy

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Time-resolved mechanisms of essential biological pathways.

Exploring cleavage activity of NSP15 using single molecule PIE-FRET

Revealing pathways of ribosome assembly and viral RNA processing with time-resolved fluorescence

A practical guide to time-resolved fluorescence microscopy and spectroscopy