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1. An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals

   https://doi.org/10.1002/adbi.202100842  

   Srinivasan, Prasanna; Griffin, Nicole_M; Thakur, DhananjayP; Joshi, PradeepM; Nguyen‐Le, Alex; McCotter, Sean; Jain, Akshar; Saeidi, Mitra; Kulkarni, Prajakta; Eisdorfer, Jaclyn_T; et al (November 2021, Advanced Biology)

   Abstract Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long‐term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage‐gated luciferase coupling the functionalities of the Ciona voltage‐sensing domain (VSD) and bacterial luciferase, luxAB. When co‐expressed with the luciferin‐producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several‐fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons ofCaenorhabditis elegansis expressed. Using the compressed sensing approach, the electropharingeogram of theC. eleganspharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition. 

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2. Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology

   https://doi.org/10.1002/nbm.4712  

   Gilad, Assaf_A; Bar‐Shir, Amnon; Bricco, Alexander_R; Mohanta, Zinia; McMahon, Michael_T (February 2022, NMR in Biomedicine)

   At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio‐organic molecules. However, later, metal‐based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal‐free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein‐based CEST agents, review the rationalization of enzyme‐substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein‐based CEST contrast agents. 

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3. A Genetically Encoded RNA Photosensitizer for Targeted Cell Regulation

   https://doi.org/10.1002/anie.202010106  

   Ren, Kewei; Keshri, Puspam; Wu, Rigumula; Sun, Zhining; Yu, Qikun; Tian, Qian; Zhao, Bin; Bagheri, Yousef; Xie, Yiwen; You, Mingxu (October 2020, Angewandte Chemie International Edition)

   Abstract Genetically encoded RNA devices have emerged for various cellular applications in imaging and biosensing, but their functions as precise regulators in living systems are still limited. Inspired by protein photosensitizers, we propose here a genetically encoded RNA aptamer based photosensitizer (GRAP). Upon illumination, the RNA photosensitizer can controllably generate reactive oxygen species for targeted cell regulation. The GRAP system can be selectively activated by endogenous stimuli and light of different wavelengths. Compared with their protein analogues, GRAP is highly programmable and exhibits reduced off‐target effects. These results indicate that GRAP enables efficient noninvasive target cell ablation with high temporal and spatial precision. This new RNA regulator system will be widely used for optogenetics, targeted cell ablation, subcellular manipulation, and imaging. 

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4. Targeted RNA condensation in living cells via genetically encodable triplet repeat tags

   https://doi.org/10.1093/nar/gkad621  

   Xue, Zhaolin; Ren, Kewei; Wu, Rigumula; Sun, Zhining; Zheng, Ru; Tian, Qian; Ali, Ahsan Ausaf; Mi, Lan; You, Mingxu (July 2023, Nucleic Acids Research)

   Abstract Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid–liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered in vitro and in living cells. Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA–protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic biology studies. 

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5. Recording of cellular physiological histories along optically readable self-assembling protein chains

   https://doi.org/10.1038/s41587-022-01586-7  

   Linghu, Changyang; An, Bobae; Shpokayte, Monika; Celiker, Orhan T.; Shmoel, Nava; Zhang, Ruihan; Zhang, Chi; Park, Demian; Park, Won Min; Ramirez, Steve; et al (January 2023, Nature Biotechnology)

   Abstract Observing cellular physiological histories is key to understanding normal and disease-related processes. Here we describe expression recording islands—a fully genetically encoded approach that enables both continual digital recording of biological information within cells and subsequent high-throughput readout in fixed cells. The information is stored in growing intracellular protein chains made of self-assembling subunits, human-designed filament-forming proteins bearing different epitope tags that each correspond to a different cellular state or function (for example, gene expression downstream of neural activity or pharmacological exposure), allowing the physiological history to be read out along the ordered subunits of protein chains with conventional optical microscopy. We use expression recording islands to record gene expression timecourse downstream of specific pharmacological and physiological stimuli in cultured neurons and in living mouse brain, with a time resolution of a fraction of a day, over periods of days to weeks. 

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