SEQUENCE SELECTIVITY OF ACRIDINIUM ESTER-LABELED PROBES

THE ULTIMATE GOAL OF THIS PROJECT IS TO DEVELOP A DNA PROBE-BASED CHEMILUMINESCENT HOMOGENEOUS ASSAY SYSTEM FOR THE RAPID, SIMPLE, AND SENSITIVE DETECTION OF INFECTIOUS DISEASE ORGANISMS IN CLINICAL SAMPLES. THE HYDROLYSIS AND SUBSEQUENT LOSS OF CHEMILUMINESCENCE OF THE ACRIDINIUM ESTERATTACHED TO A DNA PROBE HAS BEEN SHOWN TO PROCEED AT A SUBSTANTIALLY REDUCED RATE WHEN THE PROBE IS HYBRIDIZED TO ITS TARGET NUCLEIC ACID AS COMPARED TO THE UNHYBRIDIZED PROBE. THE SPECIFIC AIM OF THIS RESEARCH IS TO DETERMINE THE OPTIMAL SITE OF ACRIDINIUM ESTER ATTACHMENT WITH RESPECT TO NUCLEOTIDE SEQUENCE AND LOCATION WITHIN THE OLIGOMER TO YIELD A MAXIMUM HYDROLYSIS RATE DIFFERENTIAL BETWEEN HYBRIDIZED AND UNHYBRIDIZED OLIGOMERS. THESE PARAMETERS WILL BE STUDIED IN A SERIES OF DEFINED-SEQUENCE OLIGONUCLEOTIDES LABELED WITH ACRIDINIUM ESTER, USING KINETIC ANALYSIS OF THE HYDROLYSIS EVENT AND DETERMINATION OF THERMAL STABILITY OF THE HYBRID COMPLEX. THE DATA WILL BE USED TO REFINE AND OPTIMIZE A CHEMILUMINESCENT HOMOGENEOUS ASSAY SYSTEM BASED ON THIS DIFFERENTIAL HYDROLYSIS RATE PHENOMENON. SUCH AN ASSAY SYSTEM SHOULD SIGNIFICANTLY IMPROVE THE SPEED AND SIMPLICITYWITH WHICH INFECTIOUS DISEASES ARE CURRENTLY BEING DIAGNOSED.