Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD.

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer-ligand complex with a remarkable set of properties: Its unprecedented KD of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

Artificial RNA tags that tightly bind fluorogenic ligands have many RNA imaging and RNA-protein biomolecular purification applications. Here, we report and structurally characterize a very small (20-nt) biologically compatible G-quadruplex based aptamer that can be inserted into commonly found GNRA tetraloops. This aptamer binds its fluorogenic ligand with an unprecedented picomolar binding affinity and is very stable against thermal and chemical insults. As the ligand can be modified to include biotin, this RNA tag can also be bound to streptavidin magnetic beads. After washing, tagged RNA can be cleanly eluted by exposing the beads to intense green light, which photobleaches the bound fluorogenic ligand, triggering the release of the bound RNA complex.

An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging.

The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging. These small RNA tags bind to their fluorogenic ligands resulting in significant fluorescent enhancement, leading to a molar brightness comparable to or exceeding that of fluorescent proteins. In the past decade, multiple light-up RNA aptamer systems have been isolated that bind to a broad range of ligands involving several distinct mechanisms of fluorogenicity. This review discusses the selection methods used to isolate fluorogenic RNA aptamers. More than seventy fluorogenic aptamer:ligand pairs are evaluated using objective parameters (e.g., molar brightness, binding affinity, fluorophore exchange capabilities and other details). General guidelines for choosing fluorescent RNA tools, with an emphasis on single-molecule detection and multi-colour imaging applications are provided. Lastly the importance of global standards for evaluating fluorogenic RNA aptamer systems is discussed.

First case of Coxiella burnetii infection in Palaemonetes sinensis cultured in Liaoning, China.

The Palaemonetes sinensis aquaculture industry in Panjin City, Liaoning Province, China, experienced heavy losses in October 2018. Morbidity of cultured shrimp reached 50% and was characterized by cloudiness of muscle and the gradual spread of disease within the population. When the infection was mild, histopathological examinations revealed that the muscle cells contained a considerable number of microorganisms. In extreme cases, the structure of the hepatopancreatic glandular and muscle fiber was obscured or even vanished. Electron microscope observations revealed the presence of granular cytoplasmic inclusions in cells from hepatopancreas and muscle tissues. The 16S rDNA sequence of the intracytoplasmic organism was 94.7% identity to that of Coxiella burnetii. This is the first report of infection by C. burnetii in P. sinensis.

Metschnikowia bicuspidata isolated from milky diseased Eriocheir sinensis: Phenotypic and genetic characterization, antifungal susceptibility and challenge models.

A severe disease occurred in farmed Eriocheir sinensis characterized by milky liquid accumulation in the pectoral cavity, in the province of Liaoning, China, during October 2018-April 2019. Diseased crabs moved sluggishly, exhibited appetite loss and readily lost legs. Under the microscopic analysis, it was observed that the milky liquid contained a large number of yeastlike microorganisms (0.8-1.2 µm × 1.5-1.9 µm), which were also present in the muscle, hepatopancreas and gills. A dominant strain was isolated from the milky liquid and other tissues of diseased crabs. It grew on nutrient agar and formed 1- to 3-mm white opaque colonies, each with a protuberance in the centre. Besides, the results of TEM and SEM also demonstrate a typical multilateral budding model of the yeast clearly. We identified the strain, which we named 2EJM001, as Metschnikowia bicuspidata based on 18S rDNA, ITS and 26S rDNA sequence analyses and on its morphological, physiological and biochemical characteristics. Phylogenetic analysis revealed that 26S rDNA of 2EJM001 was clustered with M. bicuspidata (LNES0119) as reported by Bao et al. In addition, unlike Bao et al., two challenge experiments (injection and immersion) were used in this study. The results of challenge experiments show that 2EJM001 was pathogenic to E. sinensis and caused signs similar to those found in the naturally infected crabs. At the same time, the minimum inhibitory concentrations (MIC80 and MIC90 ) were determined. This study further confirms that M. bicuspidata 2EJM001 was the pathogen responsible for 'milky disease' in E. sinensis from Liaoning Province.

Photocleavable proteins that undergo fast and efficient dissociation.

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators.

Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

Optogenetic control with a photocleavable protein, PhoCl.

To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

Spying on Cells: Toward a Perfect Sleeper Agent.

Creative engineering of fluorescent proteins has yielded a variety of tools for visualization of biochemical events in vivo. In this issue of Cell Chemical Biology, To et al. (2016) describe a fluorogenic green fluorescent protein that is activated by caspase-3 activity and enables imaging of apoptosis in developing zebrafish embryos (To et al., 2016).