THE SIR SUPPLIED DR. BOLTON WITH 45G OF AS1 CELLS & 400MG SOLUTION OF DNA[U-15N]. THIS PROJECT IS AIMED AT INVESTIGATING THE BASIS FOR THE INHIBITION OF BLOOD COAGULATION BY DNA MOLECULES. AN UNDERSTANDING OF THIS INHIBITION MAY LEAD TO THE DEVELOPMENT OF IMPROVED METHODS FOR THE REGULATION OF BLOOD COAGULATION IN CLINICAL SETTINGS SUCH AS OPEN HEART SURGERY. THE DNA MOLECULE OF INTEREST IS FIFTEEN RESIDUES IN LENGTH AND IS KNOWN TO BIND AND INHIBIT THROMBIN. THE DNA MOLECULE WAS FOUND THROUGH A DIRECTED EVOLUTION SEARCH IN WHICH THE BINDING OF 1013 DISTINCT DNA MOLECULES TO THROMB THROMBIN WITH THE STRONGEST BINDING TO THROMBIN SELECTED. THE DNA MOLECULES WHICH BIND TO THROMBIN ARE REFERRED TO AS APTAMERS. THE RESEARCH PROJECT IS AIMED AT DETERMINING THE STRUCTURE OF THIS DNA MOLECULE, THE DETERMINATION OF HOW THIS MOLECULE BINDS TO THROMBIN AND THE BASIS FOR THE INHIBITION AND HOW TO USE THIS INFORMATION TO GUIDE THE DEVELOPMENT OF NEW THERAPEUTIC AGENTS. WE HAVE DETERMINED THE STRUCTURE OF THE DNA MOLECULE AND HAVE SHOWN THAT IT ADOPTS A NEW STRUCTURAL MOTIF FOR DNA WHICH MAY ALSO BE IMPORTANT IN THE STRUCTURE OF TELOMERE DNA. THIS STRUCTURE, UNLIKE THE FAMILIAR DOUBLE STRANDED DUPLES OF DNA, CAN ONLY BE ADOPTED BY A VERY LIMITED RANGE OF DNA MOLECULES. IN A SECOND STUDY WE HAVE EXAMINED THE PROPERTIES OF MODIFIED APTAMERS AND HAVE BEEN ABLE TO USE OUR STRUCTURAL INFORMATION TO PREDICT THE ABILITY OF THE MODIFIED APTAMERS TO INHIBIT BLOOD CLOTTING. IN THIS STUDY WE HAVEALSO BEEN ABLE TO IDENTITY THE BINDING SITE OF THE APTAMER TO THROMBIN. 15N LABELED DNA WILL BE ISOLATED FROM THE CELLS. THE DNA WILL BE CONVERTED INTO NUCLEOSIDES VIA THE ACTION OF NUCLEASES AND PHOSPHATASES. THE INDIVIDUAL NUCLEOSIDES WILL BE PURIFIED BY HPLC. THE PURIFIED NUCLEOSIDES WILL THEN BE CONVERTED INTO PHOSPHORAMIDITES WHICH IN TURN WILL BE USED TO SYNTHESIZE 15N LABELED APTAMERS. THE LABELED APTAMERS WILL BE COMPLEXES WITH THROMBIN AND MULTI-DIMENSIONAL NMR USED TO DETERMINE THE STRUCTURE OF THE BOUND APTAMER AS WELL AS APTAMER-THROMBIN INTERACTIONS.
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