Real-time PCR has become a crucial tool in many fields of molecular diagnostics, and many methods have been developed to monitor PCR as the reaction proceeds. The objective of this application is to examine sensing molecules such as the assembles of ZnII?DPA-attached phenylboronic acid (1.Zn) and catechol-type dyes like alizarin red S (ARS) for non-intercalator type real-time monitoring of PCR using mathematical modeling and experimental observation. The central hypothesis, which was formulated based on preliminary data, is that the 1.Zn?ARS sensor is selective towards pyrophosphate (PPi), the byproduct of DNA amplification, over the other phosphates. Different analytical evidence displayed existence multiple intertwined equilibria for the self- assembled 1.Zn-dyes sensors, yet the molecular mechanisms that underlie the phosphate sensing processes are not well understood. The long-term goal is to understand the molecular details of such very complicated self- assembly combinations that are not only limited to these examples through mathematical modeling, and to broaden this knowledge by developing novel biosensing strategies. The rationale for the proposed research is to identify unknown influences on ribonucleotide detection processes, to advance our understanding of self- assembled supramolecular host-guest thermodynamics, and to generate testable hypotheses for aptamer and drug screening procedures. To address these goals, we aim to develop a mathematical algorithm that can model the entire twenty-seven molecular intertwined interactions and reactions among the supramolecular probe, 1.Zn- ARS, and biological phosphates. We will determine the thermodynamic parameters of the interactions and reactions using the developed mathematical model, experimental design, and data fitting of the potentiometric measurements. We will then introduce optimum experimental conditions for the best real-time oligophosphate monitoring based on mathematical modeling and computer simulations. In addition, as a proof of principle, we will validate the simulated results with the experimental real-time PCR observations. This study is innovative because a sophisticated mathematical model and computer simulations will be used to investigate the reaction pathways for the formation of different guest-host bindings of 1.Zn-ARS-PPi. The computer simulations will enable the visualization of the effects of different values of binding constants, temperatures, ionic strengths, concentrations, starting pHs, etc. for any possible experimental conditions and provide appropriate directions for real-time biomolecular sensing. The proposed project is significant because, by combining the power of supramolecular self-assemblies with mathematical modeling, a better understanding of 1.Zn-ARS-PPi molecular interactions within their complex chemical networks will be achieved that can be generalized to many other complex chemical processes in pharmaceutical and biochemistry sciences to help detect and monitor analytes of medical importance.
Increased use of PCR technology for clinical and diagnostic researches has focused attention on the need for real-time monitoring of a target DNA sequence amplification, thereby avoiding time-consuming post-PCR analysis. To overcome the limitations of the presence intercalator and electrochemical real-time detection methods, which are the inhibition of PCR, preferential binding to GC-rich sequences, effects on melting curve analysis, signal disturbance by other salts in the PCR buffer, low signal resolution, and insufficient detection limit, the proposed project will utilizes mathematical modeling of a self-assembly sensor to ascertain a highly sensitive detection scheme, which indirectly probes DNA polymerization in homogeneous solution and is interference- free. The application will also provide a useful model for identification of unknown influences on ribonucleotides detection processes, advanced understanding of self-assembled supramolecular host-guest thermodynamics, and testable hypotheses to developing novel biosensing strategies.
