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1. Exploiting dCas9 fusion proteins for dynamic assembly of synthetic metabolons

   https://doi.org/10.1039/C9CC04002A  

   Berckman, Emily A.; Chen, Wilfred (July 2019, Chemical Communications)

   Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways. 

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2. A bacterial dual positive and negative selection system for dCas9 activity

   https://doi.org/10.1371/journal.pone.0269270  

   Spisak, Shaun; O’Brien, Brett; Ostermeier, Marc (June 2022, PLOS ONE)

   Fujii, Hodaka (Ed.)

   The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E . coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins. 

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3. Redox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems

   https://doi.org/10.1038/s41467-023-44223-w  

   Wang, Sally; Chen, Chen-Yu; Rzasa, John R.; Tsao, Chen-Yu; Li, Jinyang; VanArsdale, Eric; Kim, Eunkyoung; Zakaria, Fauziah Rahma; Payne, Gregory F.; Bentley, William E. (December 2023, Nature Communications)

   Abstract Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity.E. coli’s stress response regulon,oxyRS, is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology. 

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4. Simultaneous Cycloadditions in the Solid State via Supramolecular Assembly

   https://doi.org/10.1002/anie.202415567  

   Juneja, Navkiran; George, III, Gary_C; Hutchins, Kristin_M (November 2024, Angewandte Chemie International Edition)

   Abstract Chemical reactions conducted in the solid phase (specifically, crystalline) are much less numerous than solution reactions, primarily due to reduced motion, flexibility, and reactivity. The main advantage of crystalline‐state transformations is that reactant molecules can be designed to self‐assemble into specific spatial arrangements, often leading to high control over product regiochemistry and/or stereochemistry. In crystalline‐phase transformations, typically only one type of reaction occurs, and a sacrificial template molecule is frequently used to facilitate self‐assembly, similar to a catalyst or enzyme. Here, we demonstrate the first system designed to undergo two chemically unique and orthogonal cycloaddition reactions simultaneously within a single crystalline solid. Well‐controlled supramolecular self‐assembly of two molecules containing different reactive moieties affords orthogonal reactivity without use of a sacrificial template. Using only UV light, the simultaneous [2+2] and [4+4] cycloadditions are achieved regiospecifically, stereospecifically, and products are obtained in high yield, whereas a simultaneous solution‐state reaction affords a mixture of isomers in low yield. Application of dually‐reactive systems toward (supra)molecular solar thermal storage materials is also discussed. This work demonstrates fundamental chemical approaches for orthogonal reactivity in the crystalline state and highlights the complexity and reversibility that can be achieved with supramolecular design. 

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5. Site-Search Process for Synaptic Protein-DNA Complexes

   https://doi.org/10.3390/ijms23010212  

   Vemulapalli, Sridhar; Hashemi, Mohtadin; Lyubchenko, Yuri L. (January 2022, International Journal of Molecular Sciences)

   The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems. 

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