Charged with DNA AsSALT

This summer, Abby Bull, Savannah Miller, and I (Sarah Hansen) are doing biophysics research with Dr. Andresen (when we aren’t Gettypeding, that is). Although our individual projects vary, all of our research is related to the electrostatic properties of DNA. Our lab is interdisciplinary because we use the laws of physics to explain biological and chemical systems. Here is an in-depth look at our research:

IMG_20150723_161035 IMG_20150723_161627

 

 

 

 

 

 

 

 

 

 

Abby:

I am a rising Senior in the Physics department with a minor in Mathematics and have just returned from a semester abroad in Australia at the University of Melbourne. When I first came to Gettysburg College, I knew I wanted to major in physics but I was also very interested in the combination of the sciences to solve complex problems. After a few semesters, I discovered the field of biophysics by taking a course lead by Professor Andresen along with Professor Frey in the chemistry department. To further my knowledge of biophysics, I worked in Professor Andresen’s lab last summer on work similar to Sarah’s in which I investigated the electrostatics of DNA in ion competition between monovalent and trivalent cations.

If anyone has seen the chick flick Chasing Liberty, where Mandy Moore plays the president’s daughter and runs around Europe for two weeks with a secret secret agent (as in she didn’t know he was a secret agent), then I have the perfect analogy for the work I do in the lab. In one scene, Mandy Moore is with two guys and they want to hug. The secret agent is put off from this because he doesn’t want to be unmanly and hug another guy. The solution was that Mandy Moore would be in the middle of the hug and act as a “chickie buffer (that) negates the potential for man-touching-man discomfort”. The chickie buffer is what we are trying to understand in this lab. It is the conditions that exist around DNA, nucleosomes or any other highly charged macromolecules that allow them to get so close together even though they are so repulsed by one another electrostatically.

This summer I am continuing my work with electrostatics, but now I will hopefully be studying the electrostatics of nucleosomes, the first packing structure of DNA. Because I came to campus late from Australia, I have only just begun my research project. I currently have nucleosomes that are about three-quarters of the way purified that I will then expose to different ions in different concentrations to see how they react. We hope to better understand the electrostatic conditions that nucleosomes are in when they are in solution or aggregated and what causes the condensation or reabsorption of nucleosomes in solution.

Gel electrophoresis of chromatin in solution to determine the length of DNA in the nucleosomes after digestion with micrococcal nuclease. This and other methods were utilized to purify and test the nucleosomes to determine if they were adequate to use in electrostatic experiments.

Gel electrophoresis of chromatin in solution to determine the length of DNA in the nucleosomes after digestion with micrococcal nuclease. This and other methods were utilized to purify and test the nucleosomes to determine if they were adequate to use in electrostatic experiments.

Savannah:

I am a rising Junior majoring in Biochemistry and Molecular Biology and minoring in Physics. My project is a collaboration with Professor Thompson in the chemistry and is focusing on the interaction of ions with polymer coated gold nanoparticles. Gold nanoparticles are exactly what they sound like, gold particles with diameters on the nanoscale (10^-9 m). They have unique properties that make them ideal for many biomedical processes such as imaging, drug delivery, other medical therapies. They are made using a stabilizer, in this case the positively charged organic molecule CTAB, which prevents the gold from aggregating and falling out of solution. The CTAB on the surface is in equilibrium with the surrounding, which makes it impossible to isolate the gold nanoparticles without some amount of free CTAB in the solution. To rectify this, a layer of polymer can be deposited which deprives the CTAB of contact with the surrounding solution and stops the equilibrium. The polymer itself has an equilibrium constant so high that virtually none of it is exchanged. The polymer we used was Polystyrene Sulfonate (PSS) a negatively charged, sulfur containing polymer. I coated a solution of CTAB nanoparticles with the PSS polymer and then characterized them with UV-vis spectroscopy, Dynamic Light Scattering (DLS), and Zeta Potential. From this data, we can find the concentration, polydispersity, hydrodynamic radius, and the surface charge of the nanoparticle.

After coating the nanoparticles and characterizing them, I dialyzed them against different salt buffers and then against water. I characterized them again and ran both the dialysis flow through and the resultant nanoparticles through Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). ICP-OES has an argon flame that excites the electrons in each element in solution resulting in an optical emission when the electron falls back to its original state. By making calibration solutions, the machine can match the beam intensity with the concentrations of certain elements. I ran dialysis comparing sodium to potassium and found that the ion to sulfur ratio was roughly 1:1 in both cases. I noticed that the hydrodynamic radius in both cases changed based on the solution it was in. In the low salt it was around 60 nm, in the 10 mM salt it was around 50 nm, and in the pure water it was around 70 nm. Since the amount of PSS per NP is relatively unchanging, this indicates that the PSS is changing its configuration. To investigate this further, I am now doing a dialysis with a range of sodium concentrations from 5 mM to 20 mM. I am going abroad in the fall, to Lancaster University in England, but I am looking forward to continuing this research in the spring semester!

ICP-OES

ICP-OES

Sarah:

I am a rising junior, and am majoring in Physics and minoring in Math and Chemistry. My favorite aspect about biophysical research is being able to combine topics across disciplines in order to explain biological systems.  As many know from biology, the DNA structure is held together by a phosphate backbone. And as many know from chemistry, phosphate (PO43-) is negatively charged. Physics, and more specifically, electrostatics, dictates that like charges repel. Yet in our bodies, DNA is able to densely condense. In order for DNA to condense, the electrostatic repulsion generated by neighboring phosphate molecules must be overcome. In vivo, this is mediated by histone proteins, which are positively charged. Though, the specific mechanisms behind DNA-ion interactions are unknown. Many theories have been postulated to explain DNA-ion interactions, though no concrete theory has been proven to explain the system.

My project focuses on the competitive binding of hexamminecobalt(III) and magnesium to DNA as the concentration of magnesium increases and DNA-DNA spacing decreases. Hexamminecobalt(III) is trivalent, whereas magnesium ions are divalent, and it has been shown experimentally that trivalent ions are necessary to condense DNA. I was able to use Inductively Coupled Plasma – Atomic Emission Spectra to determine the amount of cobalt, magnesium, and phosphorus in my differing concentration and spacing samples, and determine the charge neutralized by cobalt and magnesium in each one. I found that as the magnesium concentration increased, more charge was neutralized by DNA, and as the DNA spacing decreased, more charge was neutralized by hexamminecobalt(III). Both of these results seem probable given the properties of the system. More recently, I have been using Matlab and Delphi to compare the theoretical Poisson-Boltzmann equation to my experimental results. This involves generating a three-dimensional hexagonal array of DNA strands to simulate the experimental conditions, as well as adjusting boundary conditions, ion valences and concentrations, and other factors to match the theoretical and experimental results.

Hexagonal array of 19 DNA helices created with Matlab.

Hexagonal array of 19 DNA helices created with Matlab.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s