The goal of this project is to develop nonisotopic detection systems based on labeled nucleotide-substituted RNA probes that can be detected in gel and in the presence of denaturants. This technology will have direct application to ultrasensitive nonisotopic ribonuclease protection assays and nucleic acid-based diagnostics. Novel labeled nucleotides will be synthesized that are capable of enhancing a luminescent-generating reaction. The labeled moities used will be small, thus allowing incorporation into nucleic acid probes that efficiently hybridize to target sequences. Nonisotopic detection is plagued by low signal/noise ratios, with background showing up well before the maximum specific signal from the probe(s) is reached. The nonisotopic detection methodologies developed through this project circumvent the two main sources of background; nonspecific binding of the probe and secondary detection components to a solid support. The solution hybridization of a ribonuclease protection assay, and the labeled nucleotide being integral to the probe, completely eliminates these background sources. This combination will allow for unrivaled nonisotopic detection sensitivity which will substantially exceed the sensitivity of 32P-based detection. A further application will be developed that will allow detection on a labeled probe-derivatized solid support directly from a cell lysate - taking advantage of the denaturant-resistant activity of the labeled nucleotides. Homogeneous assays that function under these denaturing conditions will enable transfer of RNA detection technology into a clinical setting.
The ultrasensitive nonisotopic detection methodologies developed will have wide application in molecular biology- research techniques, including ribonuclease protection assays, DNA sequencing, Southern blots, and Northern blots. The methodologies developed will also be adapted to a rapid nucleic acid-based diagnostic format that would be amenable to point-of-care testing.
