Improved Modeling of RNA/DNA Hybridization

DESCRIPTION (provided by applicant): The human genome encodes approximately 30,000 genes, and alternative splicing results in approximately 100,000 different RNAs in the cell. Genomic discoveries for disease susceptibility require thermodynamic knowledge in RNA-DNA interactions to create optimal assays using primers and probes. The overall, long-term mission of DNA Software Inc. is to facilitate genomic investigations with software that incorporates state-of-the-art knowledge of thermodynamics and kinetics of nucleic acid folding and hybridization. To enhance our existing Oligonucleotide Modeling Platform (OMP) platform, we propose to measure a complete thermodynamic i parameter database for RNA-DNA hybridization to facilitate the next step in the Human Genome Project, RNA expression assays. Most of the needed RNA-DNA nearest-neighbor parameters have never been measured. This proposal will address this gap in knowledge by performing a systematic series of measurements, appropriate data analysis and theoretical modeling. The parameters will be incorporated into OMP's database to improve the accuracy of RNA-DNA hybridization predictions. Completion of an RNA-DNA thermodynamic database will require approximately 320 new UV thermal denaturation measurements in addition to those already in the literature; thus, we are pursuing a fast-track proposal. In Phase I, we will determine the thermodynamics of 84 sequences to improve the existing match RNA-DNA parameters under various salt conditions, and perform thermal denaturation measurements on a sampling of other motifs in RNA-DNA heteroduplexes including internal single mismatches, terminal mismatches and dangling ends. These initial results will provide guidelines for the design of over 250 measurements in phase II, which will provide a complete database for mismatches, terminal mismatches, dangling ends, as well as bulges, internal loops and Mg2+ concentration dependence. Also in phase II, we will incorporate all of these parameters into our algorithms, create GUIs to extend OMP's capabilities and perform experimental validations. These accurate probe/primer designs will facilitate a variety of biotechnologies, such as gene-specific reverse-transcription PCR, cRNA expression profiling, RNA antisense technology, in situ hybridization of mRNA, Northern blot analysis, and other RNA detection methods. This work will also allow for improved detection assays for bacterial and RNA virus pathogens, which are of considerable interest for national health and biodefense.