This research proposal has the goal of measuring the rate of reaction of DNA strands with DNA or RNA to form a duplex structure, building a mathematical model for determining the rate constant of that reaction, and incorporating that model into existing software for design and simulation of oligonucleotide reactions. In phase I the rate of reaction will be measured at a single buffer condition for a series of DNA/DNA reactions with oligos designed not to have secondary structure. These oligos will vary by length, sequence, and G/C content and will have their reaction rates determined over a range of temperatures from about 15 ?C to the melting temperature. Based on preliminary studies, the dissociation rates for similar reactions have been shown to follow the Arrhenius rate equation. Mathematical models will be constructed for the rate behavior of these simple model reactions. The models will be tested and validated and the testing results will be used to improve the models. The mathematical models will then be incorporated into a spreadsheet based rate calculator that incorporates a differential equation solver and will compute reaction rates and concentrations as a function of time for oligos with no secondary structure and at the buffer conditions of phase I. In phase II the constraints from phase I on duplex hybridization reactions will be removed. In particular, the restriction of no competing secondary structures will be lifted and reaction rates will be determined for oligos pairs with secondary structure intentionally built in. Oligo pairs similar to those in phase I will be modified so that one of the strands has a controlled secondary structure, which will be modified in a systematic way. An analysis of the reaction rates of these new hairpin structures and comparison with the similar duplexes without hairpins will facilitate the elaboration of the mathematical model to include secondary structure. In addition to hairpins with small 3 base loops, molecular beacons will be used to model the effect of secondary structure in oligos with larger loops. The effect of coaxial stacking on rates will be considered for cases where two shorter oligos bind to a longer target with or without a short gap between them. In addition, phase II will also examine DNA/RNA systems, similar to the phase I DNA/DNA studies. Buffer composition has a significant impact on the reaction rate as shown in preliminary studies. A systematic study of the effect of buffer concentration on DNA/DNA reactions and DNA/RNA reaction will be carried out for sodium ion concentrations in the range of 0.1 to 1.01 M and magnesium ion concentrations in the range of 0 to 6 mM. The rate data from both phases will then be accumulated into a mathematical model which will predict the rate of reaction and oligo concentrations of all species in the reaction and will allow for all variable used in the study. This mathematical model will be incorporated as a module in our oligonucleotide modeling platform (OMP) software package. The software will then be capable predict all possible structures (e.g. monomers, self-dimers, heterodimers) in a reaction and their concentration over time, based on both kinetics and thermodynamics principles.
This project will improve a nucleic acid hybridization simulation and design tool for biochemical by adding kinetic algorithms to the pre-existing thermodynamics. This will enable researchers to develop diagnostic and therapeutic assays, while taking the time-component of a reaction into account and avoiding the formation of undesirable intermediate structures. The specific public health gains from this research will be in the areas of improved methods for analysis of genetic disorders developed with this software, while reducing false positive and false negative outcomes, and improvements in the design of gene therapies.
