Kaylee Sill (McElroy) and Dr. Robert Davis, Physics and Astronomy
Determining properties of DNA and RNA, such as conductivity of the strands, could be essential in the development of bioelectronics. Both DNA and RNA have pi bonds, which form when an electron is ‘shared’ between multiple atoms. The electrons forming pi bonds may be able to flow through a DNA strand much like electrons flow through wires when a voltage difference is introduced across the ends of the DNA strand. Some of the major difficulties in doing this for other researchers have been laying the stand across the electrodes and characterizing the connection between the electrode and the DNA. If the connection between the DNA and the electrode does not allow electrons to flow from one to the other, nothing can really be said about the conductivity of DNA.
The first step of my experiment was to learn how to use an Atomic Force Microscope (AFM) to image DNA. An AFM is able to show the topography of a surface by dragging a very small tip (about 5nm wide) across the surface. A laser reflects off the top of the tip into a detector that sends laser position information to a computer (Fig. 1). The computer then interprets the laser position as a height and displays this information on a screen.
I also had to learn how to make samples of DNA to look at with the AFM. These samples were prepared on mica, a very flat surface with layers that can be taken off one at a time. Both mica and DNA are negatively charged, so to make the DNA stick to the mica, positively charged polylysine is applied to the mica and then rinsed off. This creates a surface with a positive charge so the DNA sticks to it. A small drop of DNA solution is then placed on a corner of the surface and dragged across it to coat the surface. This leaves the surface covered with a thin layer of DNA solution.
I learned DNA sample preparation and imaging from the chemistry department since researchers there already knew how to image DNA. Changing to a similar microscope in the physics lab meant that I needed to adjust to different equipment. Primarily this meant I needed to find a way to increase the concentration of DNA on mica, since the AFM I used in the physics lab had a smaller scanning area, therefore it was difficult to locate a strand of DNA to image. Eventually I was able to create a good concentration of DNA. I did this by decreasing the concentration of DNA and slowing the speed at which it was dragged across the surface.
Next I started practicing imaging with the AFM while the sample had fluid on it. Imaging in fluid is quite different than imaging in air since it is more difficult to bring the AFM tip to the surface and the angle of the laser changes when it goes through fluid. The purpose of learning to image in fluid was to be able to see DNA in a more natural environment. This would be important to understand if DNA was to be used to create any sort of circuit.
However, at this part of the experiment our lab got a sample of ribosomes and a brand new AFM. Ribosomes are made up of RNA and protein and are the part of the cell that translates messenger RNA (mRNA) into the strand of amino acids that become a protein. Hiram Conley and I began trying to image ribosomes in fluid because no pictures of ribosomes in fluid have ever been published. A process of imaging ribosomes in fluid may allow us to determine the speed that the mRNA goes through the ribosome or to determine how compressible a ribosome is. We started teaching ourselves how to use the new AFM as well. After about two and a half months of tweaking, adjusting, and sorting out quirks we finally started getting images. During this process I created a power point presentation to teach new users how to use the microscope.
Initially, we tried imaging the ribosomes just by placing them on mica and putting a few drops of buffer on top. This did not work very well. One day we were able to get the sample ready and imaging in much less time than usual. This produced nice pictures for a while, but soon they started to look like what had been seen previously. Since ribosome samples are kept frozen until they are needed, to prevent them from falling apart, we thought imaging the sample at room temperature might be causing the sample to deteriorate. To test this we imaged the samples using a cold stage, which we used to keep the sample at about 2C. This allowed us to image samples for a few hours without the ribosomes breaking apart.
The ribosome samples were so dense that we could not get an accurate height measurement. However when we diluted the sample, the ribosomes would slip around so much on the mica that it was impossible to get an image of them. In order to remedy this we tried putting polylysine on the mica. This held the ribosomes in place so they could be imaged. This technique allows for fairly good ribosome density. However, getting a consistent height measurement for all the ribosomes in the sample will require additional research.
