Investigating substrates Amplifu Red and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the colorimetric detection of DNAzyme activity localized to DNA condensates

Through the NSF Future Manufacturing research program at Pasadena City College (PCC), students engaged in authentic research to explore aspects of DNA nanotechnology and gain experience in the research process. Emphasizing the scientific method and workforce development, students collaborated with our scientific community at UCLA, UCSB and Caltech as they learned how to use the tools of synthetic biology to build nanoscale bioreactors. Toward this goal, students set out to investigate various parameters to couple a DNAzyme-catalyzed redox reaction to DNA condensates with the aim of localizing the reaction. DNAzymes, guanine-rich sequences of DNA that fold into a G4 quadruplex structure, bind hemin, and catalyze a peroxidation reaction, were formed in vitro and used to catalyze a colorimetric redox reaction. Substrates ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and Amplifu Red were explored for their ability to ‘turn on’ or change color when oxidized by hydrogen peroxide in the presence of the peroxidase-like DNAzyme. In efforts to compartmentalize this reaction, the sequence for the G4 quadruplex was extended from one arm of a fluorescent 4-armed DNA nanostar, which contained either 15, 20, or 25 base pairs per arm and palindromic sticky ends. Upon annealing the DNA strands to form 4-armed DNA nanostars, with one of the strands containing the G4 sequence, the folded G4 quadruplex was tested for its ability to catalyze colorimetric peroxidation localized to DNA condensates. Students made important choices regarding the concentration of DNAzyme that would result in observable color change when localized to condensates; they carefully studied buffer compatibility between peroxidation and condensate formation; they tested two fluorogenic substrates in DNAzyme-catalyzed peroxidation, ABTS and Amplifu Red; and they meticulusly analyzed the results, using what they learned to inform future decisions. The results of these localization studies will be leveraged in the next steps of this research project aimed at building nanoscale bioreactors from DNA. This high-impact educational experience taught students about the iterative nature of science and the significance of exploring the literature. Through research, they learned the important higher-order skills of experimental design and effective scientific communication, facilitating their development as scientists. This synthetic biology research was translated into lessons and implemented in PCC courses and through outreach, which inspired the students taught in outreach and the PCC researchers who served as learning assistants in this equitable and accessible STEM education.