Not just a smiley face: Using DNA origami to solve problems in molecular biology

Lead Research Organisation: University of Cambridge
Department Name: Pharmacology

Abstract

The shape of the DNA molecule, a long, string-like double-helical structure is one of the iconic images of modern science. The reason that DNA has this shape is due to the chemicals that make up its structure and which in turn provide an organism's genetic code. A feature of the structure of DNA is that it has recently been shown that it is possible to split the double-helix into two, and long lengths of the resulting 'single-stranded' DNA can be folded into different shapes, producing tiny structures. This technique is called 'DNA origami'. We want to use biotechnology make DNA origami structures, and design them in such a way that we can use them to study two problems that have proved intractable using other experimental methods. We will use a very high resolution 'atomic force microscope' to look at the structures (they are very small - 725 million of them would fit on a 1.5 mm diameter pin-head). The first project is to look at the way that proteins that regulate cells' behaviour find specific points on DNA itself, which they normally identify to turn cell functions on and off. How they do this is puzzling because the proteins are very small and the DNA is very long. The second question concerns how different molecules 'talk' to each other inside a cell in order to produce signals to control of a cell's behaviour. So, we will address this by attaching all of the members of the signalling molecule family to DNA origami tiles in different patterns to show how the proximity of one member to that of another may be influential.

Technical Summary

We will design and produce DNA origami scaffolds in order to produce nano-environments to study two problems using conventional and fast-scan atomic force microscopy (AFM). In the first we will produce scaffolds that bear sequences of DANA that bear recognition sites for DNA-binding proteins. We will use these to quantify the dynamics and kinetics of protein interaction and target location on the DNA. This topic has been of interest for many years, but the data so far available remains inconclusive. The second set of experiments also addresses a question that has proved difficult to resolve using other methods, namely the spatial relationship between the differing elements of the adenylyl cyclase (AC)/protein kinase A (PKA)/phosphodiesterase (PDE) signalling system. The successful operation of this system requires correct spatial arrangement of the different components to allow, first, cAMP to be generated, then to allow it to interact with PKA, and finally, PDE must be in appropriate proximity to allow regulated degradation of cAMP. The exact geometrical relation between these components still is unknown. We will generate origami scaffolds, and attach each of the elements of the AC/PKA/PDE system in differing orientations and examine the role of orientation on activation. We will do this using fast-scan AFM. To control the initiation of the signalling cascade (which will be rapid), we will used 'caged' nucleotides. To make this possible we are collaborating with the microscope manufacturer Bruker AXS, and with them we will design build and test a flash photolysis unit using laser light, which can be used in conjunction with their fast-scanning microscope.

Planned Impact

Outside the immediate scientific communities interested in cell signalling the research will impact upon:

Users of atomic force microscopy (a very significant majority of who are in the industrial sector) who will be able to take advantage of the technical development in instrumentation that we will develop in collaboration with our industrial partner, Bruker AXS. This will be commercially advantageous both for Bruker and in terms of application and productivity to industry.

The part of the project based upon cell-signalling is important because it applies directly to mechanisms that are the target of drugs. The signalling system we will study is activated by receptors, but a puzzle is how different receptors can activate the same sort of signalling pathway in the same cell, and yet produce differing effects with in that cell. Part of this seems to be due to the physical arrangement of constituents of the signalling pathways within the cell, and that is the question we address. Clarifying this there fore has significant potential impact upon development of drugs with greater specificity, which is of interest to the pharmaceutical industry and ultimately to the public and society/policy throughout the world.

Similar commercial/societal arguments hold with the part of the project that concentrates upon mechanisms of interaction of proteins with DNA. There is considerable interest in manipulation of the genome for therapeutic purposes, and genes are controlled as the result of protein interactions, so the way in which te interaction takes place is of fundamental interest to parties interested in regulating genome function for therapeutic purposes.

Publications

10 25 50
 
Description In accordance with the original objectives we designed and produced DNA origami scaffolds in order to produce nano-environments to study two problems using conventional and fast-scan atomic force microscopy (AFM). The first aim, to produce scaffolds that bear sequences of DNA that bear recognition sites for DNA-binding proteins, and by doing so study the dynamics and kinetics of protein interaction and target location on the DNA has been largely achieved using both DNA sequences with a single recognition site for protein binding and sequences on parallel lengths of duplex DNA to allow the study of more complex DNA-binding proteins that require binding to the DNA to allow dimerization of the proteins in order to exert their action. We have also partly addressed the second proposed problem that involved the geometrical relation between the components of the AC/PKA/PDE signalling system and successfully shown activation PKA. This part of the project is technically more exacting than the other and so work continues.
Analysis of data continues and several publications are in the course of preparation for submission within the next few months.
We have also established a number of new collaborations over the course of the grant. These are described in the appropriate part of the submission. As above, we and our collaborators are preparing manuscripts that have arisen from a number of these projects.
Exploitation Route Yes. The data and development of techical skill will allow futrther work and development of existing and potential new collaborations
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We have presented our work in access and outreach activities both within Cambridge and at various venues throughout the UK. These have been mainly focused upon secondary schools and health charities. We have hosted a workshop on techniques in biological atomic force microscopy.
First Year Of Impact 2013
Sector Education,Manufacturing, including Industrial Biotechology
Impact Types Cultural,Societal,Economic

 
Title Research data supporting "Cartilage-like electrostatic stiffening of responsive cryogel scaffolds 
Description The following data files are provided: Summary of results: numerical values used to plot each figure in the manuscript, as well as values used to calculate each parameter when applicable. Raw macroindentation and microindentation data: the load vs. time curves obtained by spherical indentation on a universal testing machine and on an AFM. Confocal data: contains representative confocal datasets, presented as .lsm files, for each physical cryogel sample in the neutral and activated states. READ ME: A word document describing each file and its contents. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
 
Description AFM studies of archaeal DNA replication and repair mechanisms 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Fast scan and conventional atomic force microscopy studies of proteins involved in archaeal DNA replication and repair.
Collaborator Contribution Extensive molecular biology contributions. The partner specializes in mechanisms of archaeal DNA homeostasis, while we provide AFM imaging expertise.
Impact None yet
Start Year 2015
 
Description AFM studies of mechanisms of exocytosis 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution AFM studies of mechanisms of exocytosis.
Collaborator Contribution Experimental design and cell/molecular biology techniques and experimentation.
Impact Bao, H., Goldschen-Ohm, M., Jeggle, P., Chanda, B., Edwardson, J.M. and Chapman, E.R. (2016) Exocytotic fusion pores are composed of both lipids and proteins. Nat. Struct. Mol. Biol. 23, 67-73 One paper in press (Evans, C.S., He, Z., Bai, H., Lou, X., Jeggle, P., Sutton, R.B., Edwardson, J.M. and Chapman, E.R. (2016) Functional analysis of the interface between the tandem C2-domains of synaptotagmin-1. Mol. Biol. Cell)
 
Description AFM studies of platelets in vivo 
Organisation University of Cambridge
Department Department of Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution AFM experiments of platelets in vivo
Collaborator Contribution Collaborator is an expert in platelet biology
Impact None yet
Start Year 2015
 
Description DNA - protein interaction 
Organisation University of Edinburgh
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on atomic force microscopy studies on DNA-protein interaction. Edinburgh group are experts in the chemistry of DNA restriction-modification systems
Collaborator Contribution Named collaborator or Co-I on BBSRC-funded grants. Expertise on biology and chemistry of DNA restriction-modification systems and other DNA-binding proteins
Impact Non-multidisciplinary 1. D. T. F. Dryden, G. D. Davies, L. M. Powell, N. E. Murray, D. J. Ellis, T. Berge, J. M. Edwardson, and R. M. Henderson. The assembly of the EcoKI Type I DNA restriction/modification enzyme and its interaction with DNA. Biochem.Soc.Trans. 27 (4):691-696, 1999. 2. D. J. Ellis, D. T. F. Dryden, T. Berge, J. M. Edwardson, and R. M. Henderson. Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy. Nature Struct.Biol. 6:15-17, 1999. 3. T. Berge, D. J. Ellis, J. M. Edwardson, D. T. F. Dryden, and R. M. Henderson. Translocation-independent dimerisation of the EcoKI molecular machine revealed by atomic force microscopy. FASEB J. 14:A551, 2000. (Abstract) 4. T. Berge, D. J. Ellis, D. T. F. Dryden, J. M. Edwardson, and R. M. Henderson. Translocation-independent dimerization of the Eco KI endonuclease visualized by atomic force microscopy. Biophys.J. 79 (1):479-484, 2000. 5. M. D. Walkinshaw, P. Taylor, S. S. Sturrock, C. Atanasiu, T. Berge, R. M. Henderson, J. M. Edwardson, and D. T. F. Dryden. Structure of ocr from bacteriophage T7, a protein that mimics B-form DNA. Molecular Cell 9 (1):187-194, 2002. 6. N. Crampton, M. Yokokawa, D. T. F. Dryden, J. M. Edwardson, D. N. Rao, K. Takeyasu, S. H. Yoshimura, and R. M. Henderson. Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping. Proc Natl Acad Sci U.S.A 104 (31):12755-12760, 2007. 7. N. Crampton, S. Roes, D. T. F. Dryden, D. N. Rao, J. M. Edwardson, and R. M. Henderson. DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes. EMBO J. 26 (16):3815-3825, 2007. 8. K. J. Neaves, L. P. Cooper, J. H. White, S. M. Carnally, D. T. F. Dryden, J. M. Edwardson, and R. M. Henderson. Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping. Nucleic Acids Res. 37 (6):2053-2063, 2009. 9. D. T. F. Dryden, J. M. Edwardson, and R. M. Henderson. DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements. Nucleic Acids Res., 2011.
 
Description DNA Origami Technology 
Organisation University of Kyoto
Country Japan 
Sector Academic/University 
PI Contribution Access to expertise and materials involved in generation of DNA origami nanostructures
Collaborator Contribution Access to expertise and materials involved in generation of DNA origami nanostructures. Visits to Kyoto by PI and research personnel to learn technology and discuss research
Impact None yet
Start Year 2011
 
Description Fast scan AFM of lipid-protein interaction 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We have performed experiments using our Fast Scan atomic force microscope for studies of the interaction of proteins with artificial lipid bilayers and the effects of the proteins on membrane stability
Collaborator Contribution Provision of materials. Design of experiments
Impact None yet
Start Year 2014
 
Description GTPase activity and membrane curvature 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution Conducting fast scan atomic force micrpsciopy imageing to study Sar1 GTPase interaction with lipid membranes
Collaborator Contribution Extensive molecular biology contribution. The major portion of the work.
Impact Hanna, M.G. IV, Mela, I., Wang, L., Henderson, R.M., Chapman, E.R., Edwardson, J.M. and Audhya, A. (2016) Sar1 GTPase activity is regulated by membrane curvature. J. Biol. Chem. 291, 1014-1027
Start Year 2014
 
Description Mechanisms of tagging of DNA origami 
Organisation University of Kyoto
Department Institute of Advanced Energy
Country Japan 
Sector Academic/University 
PI Contribution Design of DNA origami templates
Collaborator Contribution Techniques of functionalisation of tags for DNA origami scaffold assembly
Impact None yet
Start Year 2015
 
Description Rapid-scanning atomic force microscopy 
Organisation University of Kyoto
Country Japan 
Sector Academic/University 
PI Contribution We have access to the rapid scanning atomic force microscope in Kyoto. This instrument is not availbel commercially and experimental versoins of it are confined to Japan. Initially we inintaited the collboration through PI and postdoc making research visits to Kyoto funded by BBSRC International travel grant.
Collaborator Contribution Access to rapid-scanning atomic force microscope and expertise of personnel in operating it.
Impact None of these outcomes can be described as 'multi-disciplinary' 1. N. Crampton, M. Yokokawa, D. T. F. Dryden, J. M. Edwardson, D. N. Rao, K. Takeyasu, S. H. Yoshimura, and R. M. Henderson. Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping. Proc Natl Acad Sci U.S.A 104 (31):12755-12760, 2007. 2. H. Takahashi, V. Shahin, R. M. Henderson, K. Takeyasu, and J. M. Edwardson. Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy. Biophys.J. 99 (8):2550-2558, 2010. 3. M. Yokokawa, S. M. Carnally, R. M. Henderson, K. Takeyasu, and J. M. Edwardson. Acid-sensing ion channel (ASIC) 1a undergoes a height transition in response to acidification. FEBS Lett. 584 (14):3107-3110, 2010. 4. Y. Suzuki, J. L. Gilmore, S. H. Yoshimura, R. M. Henderson, Y. L. Lyubchenko, and K. Takeyasu. Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region. Biophys.J. 101 (12):2992-2998, 2011. 5. Y. Suzuki, T. A. Goetze, D. Stroebel, D. Balasuriya, S. H. Yoshimura, R. M. Henderson, P. Paoletti, K. Takeyasu, and J. M. Edwardson. Visualization of structural changes accompanying activation of N-methyl-D-aspartate (NMDA) receptors using fast-scan atomic force microscopy imaging. J.Biol.Chem. 288 (2):778-784, 2013.
Start Year 2006
 
Description Sigma1 receptors 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution AFM studies of cellular signalling proteins
Collaborator Contribution Molecular biology and electrophysiology
Impact Paper in press
Start Year 2013
 
Description Biological AFM Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A workshop explaining techniques used in biological atomic force microscopy
Year(s) Of Engagement Activity 2015
 
Description Challenge Day (Cambridge 1) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 'Stretch' day for year 11 and 12 students who are considering applying for a biological science course at university
Year(s) Of Engagement Activity 2014
 
Description Challenge Day (Cambridge 2) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 'Stretch' day for years 11 and 12 students who are considering applying for a biological science course at university
Year(s) Of Engagement Activity 2015
 
Description Prospective Medical Students' Workshop, Cambridge 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Access event to explain the scientific basis of the undergraduate course in medicine at Cambridge University and to demonstrate how biomedical research might inform clinical practice (giving examples from our own and other labs). Promotion of the idea of medicine as a continuously developing scientific career.
Year(s) Of Engagement Activity 2015
 
Description School VIsit (Chelmsford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 'Stretch' day for year 12 students who are considering applying for a biological science course at university
Year(s) Of Engagement Activity 2014
 
Description School Visit (Belfast) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 'Stretch' day for year 12 students who are considering applying for a biological science course at university
Year(s) Of Engagement Activity 2016
 
Description School Visit (Harlow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 'Stretch' day for year 12 students who are considering applying for a biological science course at university
Year(s) Of Engagement Activity 2015