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

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.

Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs.

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as "sequential Fluorogenic RNA Imaging-Enabled Sensor" (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ~20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags.

Living systems contain various functional membraneless organelles that can segregate selective proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many synthetic compartments have been engineered in vitro and in living cells, mostly focused on protein-scaffolded systems. Herein, we introduce a nature-inspired genetically encoded RNA tag to program cellular condensate formations and recruit non-phase-transition target RNAs to achieve functional modulation. In our system, different lengths of CAG-repeat tags were tested as the self-assembled scaffold to drive multivalent condensate formation. Various selective target messenger RNAs and noncoding RNAs can be compartmentalized into these condensates. 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 regulation functions of these CAG-repeat tags on the cellular RNA localization, lifetime, RNA-protein interactions, and gene expression have also been investigated. Considering the importance of RNA condensation in both health and disease conditions, these genetically encodable modular and self-assembled tags can be potentially widely used for chemical biology and synthetic biology studies.

Genetically Encoded RNA-Based Bioluminescence Resonance Energy Transfer (BRET) Sensors.

RNA-based nanostructures and molecular devices have become popular for developing biosensors and genetic regulators. These programmable RNA nanodevices can be genetically encoded and modularly engineered to detect various cellular targets and then induce output signals, most often a fluorescence readout. Although powerful, the high reliance of fluorescence on the external excitation light raises concerns about its high background, photobleaching, and phototoxicity. Bioluminescence signals can be an ideal complementary readout for these genetically encoded RNA nanodevices. However, RNA-based real-time bioluminescent reporters have been rarely developed. In this study, we reported the first type of genetically encoded RNA-based bioluminescence resonance energy transfer (BRET) sensors that can be used for real-time target detection in living cells. By coupling a luciferase bioluminescence donor with a fluorogenic RNA-based acceptor, our BRET system can be modularly designed to image and detect various cellular analytes. We expect that this novel RNA-based bioluminescent system can be potentially used broadly in bioanalysis and nanomedicine for engineering biosensors, characterizing cellular RNA-protein interactions, and high-throughput screening or in vivo imaging.

Live-Cell Imaging of Guanosine Tetra- and Pentaphosphate (p)ppGpp with RNA-based Fluorescent Sensors*.

Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.

Biocontrol mechanism of Myxococcus xanthus B25-I-1 against Phytophthora infestans.

Phytophthora infestans is the pathogen causing potato late blight, one of the most serious diseases of potato. Myxobacteria have become a valuable biological control resource due to their preponderant abilities to produce various secondary metabolites with novel structure and remarkable biological activity. In this study, Myxococcus xanthus strain B25-I-1, which exhibited strong antagonistic activity against P. infestans, was isolated from soil sample and identified by 16S rRNA sequence analysis. The strain exhibited antagonistic activity against several species of fungus and bacteria. Analysis of the biocontrol mechanism showed that the active extract produced by strain B25-I-1 had strong inhibitory effects on mycelium and the asexual and sexual reproductive structures of P. infestans. Furthermore, these active extract decreased the content of soluble proteins and activity of the protective enzymes (PPO, POD, PAL, and SOD), increased the oxidative damage and the permeability of the cell membrane in P. infestans. All of these mechanisms might be the biocontrol mechanism of B25-I-1 against P. infestans. The active extract of strain B25-I-1 was separated by TLC and HPLC, and the components with antibiotic activity were detected by HPLC-MS. It was found that the antagonistic components of B25-I-1 contained methyl (2R)-2-azido-3-hydroxyl-2-methylpropanoate and N-(3-Amino-2-hydroxypropyl)-N-methylsulfuric diamide. The active extract significantly inhibited the infection on detached potato leaves by P. infestans, and these substances did not cause damage to the potato leaves. In conclusion, M. xanthus B25-I-1 produced active extract against P. infestans and might potentially be a candidate to develop into biological pesticides for the control of potato late blight. This study adds to the literature on the isolation and identification of active extracts from myxobacteria, and B25-I-1 in particular, for cures or treatments to potato late blight.

Paper-based fluorogenic RNA aptamer sensors for label-free detection of small molecules.

Sensors based on fluorogenic RNA aptamers have emerged in recent years. These sensors have been used for in vitro and intracellular detection of a broad range of biological and medical targets. However, the potential application of fluorogenic RNA-based sensors for point-of-care testing is still little studied. Here, we report a paper substrate-based portable fluorogenic RNA sensor system. Target detection can be simply performed by rehydration of RNA sensor-embedded filter papers. This affordable sensor system can be used for the selective, sensitive, and rapid detection of different target analytes, such as antibiotics and cellular signaling molecules. We believe that these paper-based fluorogenic RNA sensors show great potential for point-of-care testing of a wide range of targets from small molecules, nucleic acids, proteins, to various pathogens.

A Genetically Encoded RNA Photosensitizer for Targeted Cell Regulation.

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.

The Pre-Registry Commercial Driver Medical Examination: Screening Sensitivity and Certification Lengths for Two Safety-Related Medical Conditions.

OBJECTIVE: Use independent diagnostic data to analyze the screening effectiveness of the pre-Registry commercial driver medical examination (CDME) for obstructive sleep apnea (OSA), and its sensitivity for hypertension; analyze certification lengths where relevant. METHODS: CDME screening results for 1668 drivers were compared to polysomnogram diagnostic test results, and CDME screening results were evaluated for 1155 drivers with at least one insurance claim with a hypertension diagnostic code. Any CDME documentation of the medical condition was considered as detection by screening. RESULTS: CDME sensitivity was 20.7% for moderate OSA (AHI ≥ 15). While sensitivity was 77.5% for hypertension, 93.3% of drivers with Stage 3 hypertension were certified, contrary to Federal Motor Carrier Safety Administration standards. CONCLUSIONS: The pre-Registry CDME was ineffective in screening commercial drivers for OSA. Screening was better for hypertension; incorrect certifications were given to many hypertensive drivers.

Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules.

Imaging the cellular dynamics of metabolites and signaling molecules is critical for understanding various metabolism and signal transduction pathways. Genetically encoded RNA-based sensors are emerging powerful tools for this purpose. However, it was challenging to use these sensors to precisely determine the intracellular concentrations of target analytes. To solve this problem, we have recently developed ratiometric sensors using an orthogonal pair of RNA/fluorophore conjugates: Broccoli/DFHBI-1T (3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone) and DNB (dinitroaniline-binding aptamer)/SR-DN (sulforhodamine B-dinitroaniline). The cellular DNB-to-Broccoli fluorescence intensity ratio can be directly applied to quantify the target concentrations at the single-cell level. Unfortunately, due to the instability of the SR-DN dye, this ratiometric sensor is difficult to use for monitoring target dynamics. Herein, by replacing SR-DN with a stable TMR (tetramethylrhodamine)-DN dye, we developed a ratiometric sensor system based on Broccoli/DFHBI-1T and DNB/TMR-DN, which can be used for dynamic imaging in living cells. We believe these advanced genetically encoded ratiometric sensors can be widely used for intracellular studies of various target analytes.

Intracellular Imaging with Genetically Encoded RNA-based Molecular Sensors.

Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.