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Abstract RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA–RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA “invaders” that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of “anti-invader” strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials. 

Living cells regulate the dynamics of developmental events through interconnected signaling systems that activate and deactivate inert precursors. This suggests that similarly, synthetic biomaterials could be designed to develop over time by using chemical reaction networks to regulate the availability of assembling components. Here we demonstrate how the sequential activation or deactivation of distinct DNA building blocks can be modularly coordinated to form distinct populations of self-assembling polymers using a transcriptional signaling cascade of synthetic genes. Our building blocks are DNA tiles that polymerize into nanotubes, and whose assembly can be controlled by RNA molecules produced by synthetic genes that target the tile interaction domains. To achieve different RNA production rates, we use a strategy based on promoter “nicking” and strand displacement. By changing the way the genes are cascaded and the RNA levels, we demonstrate that we can obtain spatially and temporally different outcomes in nanotube assembly, including random DNA polymers, block polymers, and as well as distinct autonomous formation and dissolution of distinct polymer populations. Our work demonstrates a way to construct autonomous supramolecular materials whose properties depend on the timing of molecular instructions for self-assembly, and can be immediately extended to a variety of other nucleic acid circuits and assemblies. 

Incoherent feedforward networks exhibit the ability to generate temporal pulse behavior. However, exerting control over specific dynamic properties, such as amplitude and rise time, poses a challenge and is intricately tied to the network’s implementation. In this study, we focus on analyzing sequestration-based networks capable of exhibiting pulse behavior. By employing time-scale separation in the fast sequestration regime, we approximate the temporal dynamics of these networks. This approach allows us to establish a mapping that elucidates the impact of varying the kinetic rates and pulse specifications, including amplitude and rise time. Furthermore, we introduce a positive feedback mechanism to regulate the amplitude of the pulsing response. 

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 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. 

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions. 

Abstract We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA‐based receptors: a triplex‐forming DNA complex, an ATP‐binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling. 

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.