Ultrasensitive Nonisotopic Detection of RNA

Nonisotopic calorimetric and chemiluminescent technologies will be developed for use withribonuclease protection assays. The ribonuclease protection assay is an increasingly utilized techniquein molecular biology affording sensitive and quantitative measurement of mRNA levels. The ability ofthe assay to detect multiple mRNA levels simultaneously makes this technique a unique and powerfultool for use in research and diagnostic applications. In a ribonuclease protection assay, autoradiographyof the polyacrylamide gel-resolved products represents the endpoint of the experiment. However, allof the available nonisotopic detection protocols require blotting of nucleic acids onto a sold support priorto detection. Therefor, we propose an approach which will circumvent this requirement and allowdetection directly in the gel used to resolve the nucleic acids. Our initial studies demonstrated that invitro transcripts containing 4-thiouridine are both functional in ribonuclease protection assays and canbe detected in gel down to 1 picomole by a colormetric detection protocol using N-ethylamaleimide andbase. During phase I, we will synthesize a derivative of N-ethylmaleimide which has been reported togreatly potentiate the sensitivity of thiol detection. Furthermore, in an effort to extend the sensitivityof ribonuclease protection assays beyond that attainable with radioactivity, we will synthesize a thiolatedderivative of the chemiluminescent molecule isoluminol. This derivative will be coupled to either4-thiouridine- or 5 mercuriuridine-substituted transcripts and detected using a non-enzymatic, butcatalytic, oxidation system with molecules which can all readily diffuse in and out of a polyacrylamidegel matrix. Once we have developed a nonisotopic ribonuclease protection assay, it will be utilized witha panel of oncogene and tumor suppressor gene probes which are used extensively at Ambion for thedevelopment of single base mismatch technologies. Ultimately, the technologies which we develop willhave wide applications in molecular biology research extending beyond ribonuclease protection assays.