SBIR-STTR Award

This Phase I is a feasibility study of the design and synthesis of novel, non-intercalative dyes for nucleic acid (DNA/RNA) probes for DNA hybridization and other nucleic acid quantitation assays. In the past, radiolabels were used for detecting hybrids, but these are being replaced by non-radioactive probes such as oligonucleotide probes linked to fluorescent dyes. Fluorescent probes have been partially successful, but there remain significant problems associated with accurate fluorescent quantitation. Most fluorescent indicators are planar molecules that can intercalate between similarly flat base-pairs of nucleic acid helices, resulting in the quenchmg of fluorescence with consequent errors in quantitation, as well as loss in sensitivity of detection. We will develop new types of fluorescent dyes that are sterically hindered so as not to intercalate into the DNA helices when they are hnked to probe sequences. Three approaches wifl be taken: I . a sterically hindered fluorophore will be developed,2. a "dendritic" spacer unit will be developed to prevent intercalation of linked dyes and to allow multiple dye attachment, thus enabling signal multiplication, and 3. a fluorophore will be entrapped within a caged system, or host-$uest complex. The new fluorophores will be attached to deoxyuridine (dU) or deoxyuridine triphosphate (dUTP), whlch will be incorporated into oligonucleotide probes, the efficacy of which can be easily determined.Awardee's statement of the potential commercial applications of the research: There is an enormous potential for application of direct fluorescent-labeled nucleic acid probes for development of diagnostic kits for use in genome research, clinical diagnosis of parasitic, viral bacterial and genetic disorders, in cancer and AIDS research, and in agricultural and forensic applications. The availability of rapid, sensitive and reproducible fluorescence-based hybridization tests to replace radiolabeled probes would represent a considerable breakthrough with an almost universal applicability in nucleic acid analyses.National Cancer Institute (NCI)

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 fluoresoence. 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:a) development of a sterically hindered fluorophore;b) preparation of novel dendritic spacers for fluor attachment and fluorescence amplification; andc) 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.National Cancer Institute (NCI)