UV laser crosslinking of proteins with nucleic acids

For some years it has been known that proteins and nucleic acids can be crosslinked together by ultra-violet light. This phenomenon has been exploited in a number of studies investigating protein-nucleic acid interactions. We have further developed this approach by employing a high-powered pulsed UV laser as a radiation source. Using model protein-nucleic acid systems we have characterized a number of aspects of the crosslinking process. We have found that the optimal wavelength for crosslinking proteins to nucleic acids falls within the major absorption band of the nucleic acid. Thymidine residues appear to exhibit the greatest amount of crosslinking, followed by cytosine, which crosslinks with an efficiency that is about 100-fold less. We have found that the crosslinking process is critically dependent on the exact geometry of the protein-nucleic acid system, in such a way that only those partners that are involved in a very intimate contact undergo crosslinking. The rapid photon delivery time of the laser presents many advantages in crosslinking studies. Problems associated with photodegradation products, substrate exhaustion and general protein degradation can be avoided. Furthermore, since all of the crosslinking can be achieved in one short pulse lasting only several nanoseconds the distribution of proteins reflects an equilibrium that has been essentially frozen at a particular instant in time. As a result protein-nucleic acid contact information can be interpreted within a temporal context, leading to a more complete understanding of how a particular protein-nucleic acid complex functions.