This Phase II proposal is to continue development of new fluorescent probes for nucleic acid (DNA/RNA) hybridization and other nucleic acid quantification assays. In the past, radiolabels have been used extensively to detect hybrids, but these are being replaced by non-radioactive probes such as oligonucleotides conjugated to fluorescent dyes. So far, fluorescent probes have been partially successful, but there remain significant problems associated with accurate quantitation of fluorescence. Quenching of the fluorescence of the probe under assay conditions due to self- stacking and intercalation are unwanted phenomena that adversely affect the sensitivity and accuracy of fluorescence-based nucleic acid assays. In the Phase I feasibility studies, new fluorescent dyes were developed using three different molecular design strategies to overcome these limitations. These strategies are: a) Development of a sterically hindered fluorophore; b) Preparation of novel dendritic spacers for fluor attachment and fluorescence amplification; and c) Use of """"""""host-guest"""""""" fluor complexes. Based on very promising results, it is now proposed to design an entire class of fluorescent oligonucleotide probes for incorporation into diagnostic kits for in situ hybridization (FISH) assays, to be used to diagnose genetic defects and cancer as well as viral, bacterial and parasitic infections. Such simplified direct-labeled FISH assays would greatly advance molecular genetic applications in medicine.
There is an enormous potential for application of direct fluorescent-labeled nucleic acid probes for the development of diagnostic kits for use in human genome research, clinical diagnosis of parasitic, viral, bacterial, oncological and genetic disorders and in AIDS research. The availability of rapid, sensitive and reproducible fluorescence-based hybridization tests to replace radiolabeled probes would represent a breakthrough with an almost universal applicability in nucleic acid analysis.
