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Summer Intern Project 2012: Ruth Thompson

Project: Imaging DNA-substrate interactions by atomic force microscopy

Supervisors: Dr Bart Hoogenboom & Dr Guillaume Charras

Atomic Force Microscopy (AFM) is a method of imaging non-conducting samples with minimal treatment at the nanoscale, making it an attractive option for studying biological samples. In this instance, an oscillating sharp tip is brought into contact with the DNA sample under a buffer solution. By 'feeling' the sample surface, an image of the surface’s topography can be made.

A typical method of imaging biological molecules using AFM is by binding them to a cleaved mica substrate, which provides a negatively charged, atomically flat surface in solution. In order to bind DNA to this surface, which is also negatively charged in solution, divalent cations are typically used in the buffer to facilitate binding. Although the strength of binding relates to cationic radius, the exact nature of this interaction is unknown.

The main purpose of the project was to find whether there was a preferential orientation of double-stranded DNA (dsDNA) on the cleaved mica substrate, whose surface has a hexagonal lattice structure, by imaging DNA segments sufficiently short to form rods on the mica surface and statistically demonstrating a symmetry in their orientation. This is motivated by the similarity in length between dsDNA base pairs and the lattice spacing of the mica surface.

The initial training period involved imaging plasmid dsDNA, followed by 220bp dsDNA where optimal imaging conditions were found to be in 3 mM NiCl2, 10 mM HEPES pH 7 buffer at an adsorption concentration of approximately 2 ng/µl over a period between 18 and 24 hours. The mica surface is initially treated with 40 µl of 10 mM NiCl2 solution immediately after being cleaved with Scotch tape. The experimental software the data will be analysed with is being developed at EPFL, Lausanne. Another area of interest is the possibility of distinguishing left and right handed DNA by measuring their length. This follows from recent high resolution AFM imaging of dsDNA performed by the research group.

Figure: Imaging 220 bp dsDNA adsorbed on mica in buffer using AFM

An alternative method of binding DNA to a mica substrate is by functionalising the mica to create a positive surface charge in solution, removing the need for divalent cations. Methods for producing APTES-mica were consulted in the literature, and were attempted with varying success. Possible improvements were suggested. There was also the opportunity to image IKKγ proteins, the results of which suggested the proteins had a rod-like shape. These samples were prepared similarly to DNA, but in a buffer of 20 mM MgCl2, 25 mM TRIS, 200 mM NaCl pH 7.5 at an adsorption concentration of 10 ng/µl. Further imaging, if new samples become available, may lend support to theories of a “coiled coil” structure.

This summer project has provided invaluable laboratory experience in an interdisciplinary research environment, and will form the basis of my MSci project.