Exploring Toehold-Mediated Strand Displacement as a Strategy to Program DNAzyme-catalyzed Peroxidation Localized to DNA Condensates

DNA nanotechnology can be leveraged to engineer nanoscale biochemical reactions, and thus, revolutionize biomanufacturing. The programmability is encoded in the interactions between base pairs of the nucleic acids. Functional nanostructures can be envisioned and formed, such as DNA nanostars, whose properties can be fine-tuned by engineering the number of arms or base pairs per arm and can yield synthetic condensate structures, and DNA-based enzymes that exhibit peroxidase-like activity. For example, certain guanine-rich sequences of DNA can fold into a quadruplex structure, bind a hemin co-factor, and catalyze a peroxidation reaction in which the substrate ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) gets oxidized by hydrogen peroxide and results in a colorimetric change. Because ABTS produces a blue-green color change upon oxidation, it can be used to visually observe the peroxidation reaction taking place within the DNA condensates. In this work, peroxidase-mimicking DNAzymes were used to catalyze colorimetric peroxidation within DNA condensate compartments; and toehold-mediated strand displacement (TMSD) was explored as a strategy to program the peroxidation reaction–specifically, by unwinding the G-quadruplex structure, which would effectively turn the reaction “off”. TMSD is a method of designing a single strand of DNA with an additional overhang region, called a toehold, to oust and replace a second strand attached to the toehold-possessing target strand. The presence of complementary toeholds on both the invading strand and the target strand increases the thermodynamic probability of displacing the single DNA strand originally bound to the target. Here, TMSD was adapted for use in ‘turning off’ the DNAzyme-catalyzed peroxidation reaction, either by preventing folding or disrupting the folded structure of the DNAzyme. A displacer strand complementary to the DNAzyme/toehold region was designed and added to the reaction mixture at different time points and concentrations for this purpose. Elucidating mechanisms to unwind the G-quadruplex structure of DNAzymes has promise in treating genetic disorders caused by unregulated G4 formation in the human genome. Furthermore, DNA nanotechnology can be used to compartmentalize, functionalize, and program the release of bioactive molecules in drug delivery strategies and other synthetic biology applications, highlighting the potential of TMSD to program DNA-based bioreactors. This high-impact study, carried out as part of the NSF Future Manufacturing program at Pasadena City College in collaboration with UCLA, UCSB, and Caltech, allowed undergraduate researchers to design and conduct their own experiments within a community college setting after undergoing scientific training by graduate students and postdocs from our collaborators’ institutions. It also provided opportunities to communicate the scientific research through writing, poster presentations at national conferences, and teaching in courses and STEM outreach. The student researchers of the PCC nanostar program applied their knowledge in a classroom setting, where they taught other undergraduate students how to conduct aspects of this research in a General, Organic and Biochemistry laboratory course at PCC. This article underscores the importance of creating significant research and teaching opportunities for students as they begin their careers in STEM, impactful mentorship through undergraduate research, and the creativity involved in modern synthetic biology research and in the development of accessible and innovative science lessons.