PROJECT SUMMARY/ABSTRACT
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.
