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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. \n\nIt 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. 

Through the NSF Future Manufacturing undergraduate research program at Pasadena City College (PCC), students utilize the tools of synthetic biology to build sustainable, DNA-based materials. The manipulation of DNA enables the construction of microscopic biochemical reactors through the formation of liquid-liquid phase-separated droplets, or DNA condensates. This research investigates the potential of DNA nanostars fused with G-tetraplexes, which can bind hemin, an iron-containing porphyrin co-factor, to form a DNAzyme capable of catalyzing peroxidation reactions within single condensate layers. The in vitro component of this research was enhanced by in silico coarse-grained molecular dynamics simulations, which generated 3D models of the DNA nanostars that allowed student researchers to visualize the behavior of the structures created in the laboratory. Leveraging this computational technique, student researchers developed educational resources and modular lessons to introduce these molecular simulations to a broad student audience at PCC. The simulation programs used, oxDNA and oxView, were instrumental in making this research accessible and engaging for diverse student groups. DNA nanostar simulations were integrated into the General, Organic, and Biochemistry curriculum at PCC, as well as during outreach events such as Girls Science Day, offering students insights into DNA nanostar dynamics and potential applications of DNA-based inventions. This paper details the use of simulation programs to recreate nucleic acid-based nanostructures, advancing the field of DNA nanotechnology. Molecular simulations helped the PCC research students develop experiments that demonstrate how enzymatic activity within DNA droplets can be achieved through G4 complexing. Simulating DNA nanostars with G4s was a profound educational exercise for students, as it taught them about the powerful synergy between in silico and in vitro experimentation. Students also learned about the limitations of modeling biomolecules using computational software, and our G4 simulation results may even inspire the integration of guanine-guanine interactions into the oxDNA program. These findings underscore the significant implications of in silico modeling and structural analysis in biochemical manufacturing and industrial applications, paving the way for further innovations in programmable biomolecular systems. By developing YouTube tutorials that teach students how to carry out nucleic acid simulations on any standard computer, the exploration of DNA dynamics and molecular programming is now widely accessible to both students and educators.