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

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.

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.

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.