Practical Course Prof. Dr. Axel A. Brakhage

„Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance“
Leader: Dr. Peter Hortschansky

Date and time: 16 - 17 June 2008, from 08:30 - 18:00
Place: HKI, Dept. Molecular and Applied Microbiology, Beutenbergstr. 11a, 1st floor, room C218

Please note: The number of participants is limited to 6 students!

Summary
With the discovery of the DNA structure by Watson and Crick we started to understand that the genetic information of the cell is encrypted in DNA. By now this genetic information is available for many species due to large-scale DNA sequencing projects. The cell accesses genetic information stored in DNA through specialized proteins, which can bind to DNA. Frequently, numerous different proteins collaborate to build multicomponent complexes on top of DNA, which are required for the regulation of gene expression and the replication, modification, and repair of DNA.
To understand the complex interaction between proteins, protein complexes, and DNA several methods have been developed with the focus being to define the interaction in qualitative or quantitative terms. Historical methods that measure protein-DNA interactions like footprinting and EMSA are qualitative or semiquantitative, because they are not compatible with the fast association rates of many DNA binding proteins.
This led to the development of biophysical methods, which rely on fast optical biosensor systems for quantitative protein-DNA interaction measurements. One of the most important optical methods to monitor the interaction between biomolecules is Surface Plasmon Resonance (SPR). One molecule is immobilized on a chip surface and binding of the other molecule is detected as a change of refractive index on the chip surface in real time. Since the change in refractive index corresponds to a change in mass this method can also yield data on the stoichiometry of complexes in addition to binding kinetics.

Course topics:
· Surface Plasmon Resonance principle
· Sensor chip surface properties
· Ligand immobilization strategies
· SPR Assay Development
· Preparation of a SPR sensor chip and DNA duplex capturing
· Performing a SPR Protein-DNA interaction assay (Kinetic and Affinity Analysis)
· Sensorgram evaluation (Determination of rate and binding constants)