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The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5′-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.
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DNA Catalysis: Design, Function, and Optimization
Catalytic DNA has gained significant attention in recent decades as a highly efficient and tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization. Despite being composed of just four monomers, DNA’s complex conformational intricacies enable a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving the way for rational structural optimization. This review will summarize the latest studies on the performance and optimization of traditional DNAzymes and provide an in-depth analysis of DNAzyme hybrid catalysts and their unique and promising properties.
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- Award ID(s):
- 2247709
- PAR ID:
- 10587837
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 29
- Issue:
- 21
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 5011
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules29215011
