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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.
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Calcium-Dependent Chemiluminescence Catalyzed by a Truncated c-MYC Promoter G-Triplex DNA
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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- Award ID(s):
- 2122041
- PAR ID:
- 10598290
- Publisher / Repository:
- Molecules (MDPI)
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 29
- Issue:
- 18
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 4457
- 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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The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities.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/molecules29184457
