In this proposal Dr. Brent intends to use a novel set of artificially produced inhibitors called aptamers to investigate the structural parameters which modulate protein-protein interactions and to investigate cell cycle regulation. Aptamers are generated by inserting random sequences encoding 20 amino acids into a stable loop which normally extends out away from surface of thioredoxin. To screen this random library for fusion proteins, which interact with specific target proteins of interest, the thioredoxin proteins or """"""""prey"""""""" also contain the B112 transcription domains and the target proteins or bait lex A. Screening for interaction between prey and bait is then achieved by a modified version of the two-hybrid screen. The technology has been used to identify 14-aptamers out of 6 x 106 which interact with high affinity (Kd = 10 -7 -10 -8) to cdk2. The investigator has shown that these cdk2 aptamers block cdk2-cyclin E kinase activity when H1 is a substrate. Thus, the methodology can be used effectively to identify and isolate sequences which bind to specific proteins and inhibit their activity. In this grant the Principal Investigator proposes to use this new set of reagents to better characterize and define the function of 3 cell cycle regulators Cdc2, Cdk2, and Cdi1.
