Single Molecule Imaging

Lead Research Organisation: MRC Clinical Sciences Centre

Abstract

Single molecule microscopy has opened up new avenues leading to important discoveries on how structural dynamics correlate to the function of nucleic acids and proteins. An attractive aspect of single-molecule microscopy is that it reveals the structural dynamics of individual molecules, otherwise hidden in ensemble-averaged experiments, thereby providing direct observation of key reaction intermediates (even low populated or short lived ones) and the characterization of reaction mechanisms. Our group develops single molecule technologies to study key cellular processed such as RNA splicing, DNA replication and deamination.

Technical Summary

The overall aim of the program is to develop single-molecule and super-resolution imaging methodologies to monitor key cellular processes in real time. In recent years, cellular imaging has witnessed a remarkable revolution with the advent of single molecule spectroscopy and super-resolution microscopy. These technologies enable the direct observation of biochemical pathways in real time, and to characterize them in unprecedented detail.
Specifically, our group focuses on three main topics:
RNA Splicing is an essential step in the maturation of eukaryotic pre-mRNAs. Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous diseases such as breast, colorectal, epithelial and ovarian cancers, as well as neurodegenerative disorders such as Parkinsons and Alzheimers. The spliceosome is a large dynamic assembly of 5 small nuclear RNAs (snRNA) and over 100 proteins that catalyzes splicing. It undergoes several, highly conserved, conformational rearrangements. The active site of the spliceosome comprises two key snRNAs (U2 and U6) that have been shown to undergo splicing-related catalysis in absence of proteins. The structure and dynamics of the U2-U6 complex are thought to play critical roles in the mechanism of splicing in vivo. Using active spliceosomes in yeast cell extracts reconstituted with flurophore-labeled U6 snRNA, we explore the role of these dynamics in splicing assembly and catalysis.
The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3-hydroxyl of the growing DNA chain. However, the mechanism by which they achieve their extraordinary accuracy remains unclear. Specifically, kinetic intermediates involved in proofreading have never before been characterized. We use single-molecule approaches to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution.
The APOBEC family of enzymes comprises single-stranded ssDNA cytosine deaminases that play important roles in eliminating retroviral infectivity and somatic hypermutation (SHM). For example, APOBEC3G eliminates HIV1 infectivity by converting C?U in numerous small target motifs on the minus viral cDNA, wheras AID generates advantageous mutations in the variable region of immunoglobulin genes in B-cells that increase the affinity of antibodies for antigenes. Compared with dsDNA scanning enzymes (e.g., DNA glycosylases) that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. We investigate ssDNA scanning and motif-targeting mechanisms for Apo3G and AID using single molecule fluorescence. We address specific issues of deamination activity within the general context of ssDNA scanning mechanisms.

Publications


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Alemán EA (2014) Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle. in The journal of physical chemistry letters
Paudel B (2014) RNA folding dynamics using laser-assisted single-molecule refolding. in Methods in molecular biology (Clifton, N.J.)
 
Description Marie Sklowdoska-Curie Actions Individual Fellowships
Amount £183,454 (GBP)
Funding ID 705015 
Organisation European Commission (EC) 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 06/2016 
End 06/2018
 
Description Research Project Grant
Amount £186,209 (GBP)
Funding ID RPG-2016-214 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2016 
End 08/2019
 
Description Assembly and Folding of eukaryotic spliceosomal ribonucleoprotein complexes 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2013
 
Description Elucidating DNA replication fidelity mechanisms with single molecule microscopy 
Organisation Wayne State University
Department Department of Chemistry
Country United States of America 
Sector Academic/University 
PI Contribution Research Collaboration and Papers
Collaborator Contribution Research Collaboration and Papers
Impact Research Collaboration and Papers
Start Year 2006
 
Description Helicase Assisted Folding of Large Ribozymes 
Organisation School of Medicine Yale
Department Department of Cell Biology
Country United States of America 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2008
 
Description Imaging Human T-cell Leukaemia Virus Integration 
Organisation Imperial College London (ICL)
Department Department of Infectious Diseases and Immunity
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2013
 
Description Imaging Influenza virus assembly with single molecule resolution 
Organisation Department of Medicine
Department Section of Virology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2013
 
Description Mechanism and Dynamics of Retroviral Integration 
Organisation Imperial College London (ICL)
Department Department of Infectious Diseases and Immunity
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2013
 
Description Mechanisms of Alternative Splicing Regulation 
Organisation Swiss Federal Institute of Technology Zurich (Eidgenössische Technische Hochschule Zürich ETH)
Department Institute of Molecular Biology & Biophysics
Country Switzerland, Swiss Confederation 
Sector Public 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
 
Description RNA folding mechanisms of large RNA enzymes 
Organisation University of Zurich (Universität Zürich UZH)
Department Department of Chemistry
Country Switzerland, Swiss Confederation 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
 
Description RNA interactions in HIV-1 
Organisation University of Strasbourg
Department Structure and Reactivity or RNA
Country France, French Republic 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2010
 
Description Watching AID/Apobec scanning and deaminating DNA with single molecule resolution 
Organisation College of Letters, Arts and Sciences
Department Department of Biological Sciences
Country United States of America 
Sector Academic/University 
PI Contribution Research and Papers
Collaborator Contribution Research and Papers
Impact Research and Papers
Start Year 2009