Biomolecular Imaging

S Allen , MC Davies , CJ Roberts , SJB Tendler , PM Williams

The scanning probe microscope offers unprecedented imaging resolution. In the 1980s the LBSA pioneered the application of SPM techniques for biomolecular studies. Our success in STM imaging of proteins and DNA under ambient conditions was achieved through the development of new instrumentation, chemically characterized and validated sample preparation techniques, and novel analysis tools. Through this combination of skills we are now able to address important questions surrounding biomolecular structure and function. Using predominately AFM in concert with new chemistries and tip technologies we are studying a variety of biological structural problems. Examples of this work include 

• Studies of DNA condensation by polymers for gene therapy applications
• Structure of clamp-loader machinery in Bacillus stearothermophilus
• Development of new imaging techniques
  
  
  

Studies of DNA condensation by polymers for gene therapy applications

Successful delivery of DNA to a cell nucleus for gene therapy requires compaction of the DNA and its protection from enzymatic and chemical degradation. The traditional in vitro methods used to study these processes provide no direct link between the molecular structure of the vector complexes and their success. Molecular real-time AFM imaging in liquid permitted us to visualise the effect of DNase I on generation 4 polyamidoamine dendrimers complexed with DNA. The formation of the G4–DNA complexes was observed to provide protection to the DNA, and this protection was found to be related to the structural morphology of the complexes formed.

 

Click on the image on the right-hand side to download an AVI movie (18.6 Mb). This movie is a sequence of images acquired over a period of 15 minutes where a single plasmid is seen to unwind after one of its strands is nicked by cleavage of a DNase I molecule.

Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I
Abdelhady HG, Allen S, Davies MC, Roberts CJ, Tendler SJB, Williams PM 
Nucleic Acids Research 31 (14): 4001-4005, 2003


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Structure of clamp-loader machinery in Bacillus stearothermophilus

High-resolution AFM imaging, including the use of single-walled carbon nanotube tips, was able to reveal the structure of the clamp-loader-helicase complex of Bacillus stearothermophilus. The system shows architecture similar to the Escherichia coli counterpart.

 
DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings
Thirlway J, Turner IJ, Gibson CT, Gardiner L, Brady K, Allen S, Roberts CJ, Soultanas P 
Nucleic Acids Research 32 (10): 2977-2986, 2004


We have continued to explore the architecture of the protein components in this replication machinery, and through a combination of imaging and force measurements reveal new insight of this very biological machinery, for example,

The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA
Zhang WK, Allen S, Roberts CJ, Soultanas P 
Journal of Bacteriology 188 (15): 5487-5493, 2006

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication
Turner IJ, Scott DJ, Allen S, Roberts CJ, Soultanas P 
FEBS Letters 577 (3): 460-464, 2004



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Devlopment of new imaging techniques

 

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Academic staff

Dr S Allen - Associate Professor and Reader in Molecular Biophysics

Professor MC Davies  - Professor of Biomedical Surface Chemistry

Professor CJ Roberts  - Professor in Pharmaceutical Nanotechnology

Professor SJB Tendler  - Professor of Biophysical Chemistry

Professor PM Williams  - Chair of Theoretical Biophysics