(612a) A Mathematical Model to Reproduce Biphasic DNA Amplification Output

DNA amplification technologies are a cornerstone of molecular diagnostics. While Polymerase Chain Reaction (PCR) provides a gold standard for accuracy and sensitivity of nucleic acid detection, it requires complex laboratory equipment and trained personnel. Isothermal DNA amplification reactions that require less energy, time, and equipment than PCR have therefore become an increasingly popular alternative for molecular detection. Exponential Amplification Reaction (EXPAR) is one such popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. As these short oligonucleotide triggers can be created by specific proteins, microRNA, DNA, and RNA, amplification reactions of this type are a popular tool for molecular recognition schemes. These types of reactions also are used in DNA circuits and show promise in synthetic biology. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrates unexpected biphasic amplification and much higher DNA yield than EXPAR. This Ultrasensitive DNA Amplification Reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms. The reaction mechanisms are (i) positively cooperative multistep binding spurred by two trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. The model reproduces experimental biphasic ouput and product concentrations using reasonable kinetic parameter estimates from values reported in literature. Furthermore, we show that three of the proposed mechanisms are necessary to fully reproduce the experimental data. These mechanisms can potentially illuminate behavior of EXPAR as well as other nulceic acid amplification reactions. Our ultimate goal is to predict and control the timing, duration, and trigger concentration at the plateaus based on known DNA association and dissociation kinetics. This work is relevant for general kinetic modeling of DNA amplification reactions used in DNA circuits, as well as quantitative molecular recognition.

Acknowledgements:

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program, Discovery Award under Award No. W81XWH-17-1-0319. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. TG was partially supported by NSF grant DMS-1361240, USDA 2015-51106-23970, DARPA grant FA8750-17-C-0054, and NIH grant 1R01GM126555-01. DC acknowledges the SMART Scholarship funded by the Department of Defense (US Army).