In this Early Concept Grant for Exploratory Research, funded by the Chemical Measurement and Imaging Program of the Division of Chemistry, Professor Eisenthal from Columbia University will measure affinity constants between biomolecules by the application of nonlinear spectroscopic techniques. Affinity constants are used to characterize the strength of interactions from biomolecule to biomolecule. The new non-linear optical method responds to changes in electric charge in the solid/water interface when free biomolecules from the solution complex with biomolecules that are bound to the solid side of the interface. The proposed technique may provide a new path in the search for novel drug therapies.
We have developed a radically new method to investigate the interactions of biomolecules with other biomolecules such as genetic molecules (DNA), proteins, sequences of amino acids strung together, and drugs that are anti-cancer, antibiotic, and anti-viral. This new method developed with NSF support is based on the recognition that biomolecules typically have an electrical charge that upon reacting with other biomolecules, which are present in the solution, can generate products having a different electrical charge. We shine pulses of laser light on a water solution containing the molecules that we seek to investigate. The light that we use is of very high power( billions of watts) and short duration( one hundred thousandth of a billionth of a second) and is of one color, let us say red, and detect light that is generated by the reacting molecules, that is precisely in the blue. Such intense pulses of light enable us to directly probe the molecules of interest. We applied this technique to a reaction involving DNA. Bacteria have developed, a defense against viruses that penetrate the cell and that without a defense would destroy the bacteria. Over a very long period of time bacteria have survived by developing an enzyme, which recognizes that the DNA of the invading virus is foreign, and proceeds to bind to the viral DNA and then cleaves the DNA, which thereby kills the virus. The DNA fragments from the DNA target have different electrical charge that is revealed by the change in the intensity of the blue light. We have succeeded in observing the binding, cleaving and the effectiveness of separation of the DNA fragments in real time. The property of the enzyme to cleave DNA at a particular location on the DNA helix, has made it a valuable tool in studying recombination of DNA strands and cloning of DNA. An important feature of our laser method is that we do not attach a molecule to DNA, which other methods need to do in order to observe the biological reaction. The problem with the label is that it can affect the very interaction that is under investigation. In another project supported by NSF we demonstrated that we can measure the strength of binding of drugs to DNA using the same conceptually straight forward and simple laser technique, which makes it potentially a powerful new way to probe biological processes. We have applied this technique to measure the binding constant of the anti-cancer drug daunomycin to DNA. Being able to measure the binding strength of a drug to DNA is a key part in determining the potential effectiveness of the drug.
