Engineering and In Vivo Applications of Riboswitches.

Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.

An optimized bioluminescent substrate for non-invasive imaging in the brain.

Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons.

Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

In Vitro and In Vivo Enzyme Activity Screening via RNA-Based Fluorescent Biosensors for S-Adenosyl-l-homocysteine (SAH).

High-throughput enzyme activity screens are essential for target characterization and drug development, but few assays employ techniques or reagents that are applicable to both in vitro and live cell settings. Here, we present a class of selective and sensitive fluorescent biosensors for S-adenosyl-l-homocysteine (SAH) that provide a direct "mix and go" activity assay for methyltransferases (MTases), an enzyme class that includes several cancer therapeutic targets. Our riboswitch-based biosensors required an alternate inverted fusion design strategy, but retained full selectivity for SAH over its close structural analogue, the highly abundant methylation cofactor S-adenosyl-l-methionine (SAM). The level of ligand selectivity for these fluorescent biosensors exceeded that of commercial antibodies for SAH and proved critical to cellular applications, as we employed them to measure methylthioadenosine nucleosidase (MTAN) activity in live Escherichia coli. In particular, we were able to monitor in vivo increase of SAH levels upon chemical inhibition of MTAN using flow cytometry, which demonstrates high-throughput, single cell measurement of an enzyme activity associated with the biosynthesis of quorum sensing signal AI-2. Thus, this study presents RNA-based fluorescent biosensors as promising molecular reagents for high-throughput enzymatic assays that successfully bridge the gap between in vitro and in vivo applications.

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Medial and dorsal lateral septum involving social disruption stress-primed escalation in acid-induced writhes.

Introduction: Stress may cause prospective escalations in abdominal pain magnitude and accumbal TRPV1 expression, while central neural circuits mediating these stress effects remain unclear. Methods: Using retrograde tracing methods, we first demonstrated the existence of a medial septal-dorsal lateral septal -accumbal circuit very likely involving social disruption stress-primed escalations in acid-induced writhes and accumbal TRPV1 level. An intersectional viral strategy and virus-carrying hM3Dq and hM4Di DREADDs were, then, employed to selectively modulate GABAergic and cholinergic neuronal activity in medial and dorsal lateral septum. Results: Exciting medial septal GABAergic neuron was found to prevent social disruption stress-primed escalations in acid-induced writhes and accumbal TRPV1 and PKCε expressions. Likewise, inactivating dorsal lateral septal cholinergic neurons was also effective in abolishing these stress-primed escalations. Inactivating GABAergic neuron in non-stressed animals' medial septum was found to reproduce the stress-primed effects in causing heightened acid-induced writhes and accumbal TRPV1 and PKCε levels. Discussion: These results, taken together, prompt us to conclude that social disruption stress may produce plastic changes in a newly-identified medial septal-dorsal lateral septal-accumbal circuit. Moreover, medial septal GABAergic hypoactivity and dorsal lateral septal cholinergic hyperactivity are, at least, two likely causes reflecting such stress-produced escalations in abdominal pain magnitude and pain transduction-related protein over-expression in nucleus accumbens.

Kinase-Modulated Bioluminescent Indicators Enable Noninvasive Imaging of Drug Activity in the Brain.

Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for noninvasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. ERK KiMBI discriminates between brain-penetrant and nonpenetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging.

Bioluminescence imaging (BLI) using luciferase reporters is an indispensable method for the noninvasive visualization of cell populations and biochemical events in living animals. BLI is widely performed with preclinical rodent models to understand disease processes and evaluate potential cell- or gene-based therapies. However, in vivo BLI remains constrained by low photon production and tissue attenuation, limiting the sensitivity of reporting from small numbers of cells in deep locations and hindering its application to larger animal models. This Review highlights recent advances in the development of luciferase systems that improve the sensitivity of in vivo BLI and discusses the expanding array of biological applications.

Observer's adrenal corticosterone secretion involvement in vicarious fear conditioning.

Vicarious learning represents a far-reaching value for the survival of social animals. Adrenal hormones are known to affect many forms of learning, yet the roles of adrenal hormones in vicarious learning remain unexplored. This study was undertaken to assess whether observation-stimulated corticosterone (CORT) secretion may affect the magnitude of a vicarious fear conditioning. Mouse observers were individually subjected to an observational compartment next to the training compartment wherein three their cage-mate demonstrators received (1) 5 days of 15 randomly-scheduled footshocks (0.5 mA, 2 s in duration over a 30 min session) (G1); (2) a 30-min presentation of vanilla odors (G2); or (3) footshock delivery and vanilla odors in combination (G3). Demonstrator mice receiving G3 training session and their respective observer mice were found to exhibit greater training-induced and slightly greater observation-stimulated CORT secretion, greater vanilla odors-induced fear responses (FR) and conditioned place aversion (CPA), as compared with the observers vicariously learning from demonstrators receiving G1 or G2 sessions. Observers held in their home cages during demonstrators' trainings and those receiving null demonstrator (No Demonstrator) failed to exhibit vanilla odors-induced FR. Moreover, observers undergoing adrenalectomy (ADX) and G3 sessions exhibited lower vanilla odors-induced FR and CPA as compared to sham surgical (Sham) observers observing G3 sessions. Furthermore, systemic metyrapone injections (50 and 100 mg/kg) prior to daily vicarious G3 training session resulted in decreases in vanilla odors-induced FR and CPA magnitudes in observers. Finally, CORT (1 mg/kg)-pretreated G2 observers failed to display odors-induced FR escalation. These results, taken together, suggest that observation-stimulated CORT secretion is necessary for reliable establishment of vicarious fear conditioning in observer mice.

Social disruption-induced stress pre-exposure aggravates, while the presence of conspecifics diminishes, acetic acid-induced writhing.

RATIONALE AND OBJECTIVE: This study was undertaken to assess the modulating effects of (1) pre-exposure to repeated social disruption and (2) group testing on writhing associated with visceral pain induced by intraperitoneal administration of acetic acid. MATERIALS AND METHODS: Six consecutive days of social disruption were used to prime for stress, while group testing referred to 3 mouse cage-mates receiving the acetic acid-induced writhing test as a group. RESULTS: Social disruption-induced stress-pre-exposed mice displayed a greater number acid-induced writhes compared to mice not receiving the pre-exposure. However, mice displayed fewer acid-induced writhes in a triad group vs. individually, suggesting group-mediated writhing-reducing effects. Likewise, group testing prevented the stress pre-exposure escalation in acid-induced writhes. Additional studies revealed that the stress-pre-exposed mice had increased expression in accumbal TRPV1 receptors. Systemic (0.25 mg/kg) and bilateral intra-accumbal (0.2 ng/0.2 µl/side) administration of SB366791, a TRPV1 receptor antagonist, reliably prevented the stress pre-exposure escalation in acid-induced writhing; SB366791 treatment alone did not affect acid-induced writhing, stress pre-exposure anxiety-like behavior, or the group testing effects. Furthermore, lower neuronal activation was found in the medial septal nucleus in group vs. individual tested mice. Intra-medial septum (0.2 µg/0.5 µl) infusion with bicuculline, a GABAA receptor antagonist, effectively prevented group-mediated writhing-reducing effects, but not individual acid-induced writhing effects. CONCLUSIONS: These findings suggest that social disruption-induced stress pre-exposure may upregulate accumbal TRPV1 receptor expression and consequently aggravate acid-induced writhing. Group testing prevents such stress pre-exposure escalation of acid-induced writhing most likely by strengthening the GABAergic inhibition on local neural activity in the medial septum.

RNA-based fluorescent biosensors for live cell imaging of small molecules and RNAs.

Genetically encodable fluorescent biosensors provide spatiotemporal information on their target analytes in a label-free manner, which has enabled the study of cell biology and signaling in living cells. Over the past three decades, fueled by the development of a wide palette of fluorescent proteins, protein-based fluorescent biosensors against a broad array of targets have been developed. Recently, with the development of fluorogenic RNA aptamer-dye pairs that function in live cells, RNA-based fluorescent (RBF) biosensors have emerged as a complementary class of biosensors. Here we review the current state-of-the-art for fluorogenic RNA aptamers and RBF biosensors for imaging small molecules and RNAs, and highlight some emerging opportunities.

Synthetic Biology of Small RNAs and Riboswitches.

In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in cis based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.

Systemic anti-hepatocyte growth factor monoclonal antibody therapy induces the regression of intracranial glioma xenografts.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor Met are involved in the initiation, progression, and metastasis of numerous systemic and central nervous system tumors. Thus, an anti-HGF monoclonal antibody (mAb) capable of blocking the HGF-Met interaction could have broad applicability in cancer therapy. EXPERIMENTAL DESIGN: An anti-HGF mAb L2G7 that blocks binding of HGF to Met was generated by hybridoma technology, and its ability to inhibit the various biological activities of HGF was measured by in vitro assays. The ability of L2G7 to inhibit the growth of tumors was determined by establishing s.c. and intracranial xenografts of human U87 and U118 glioma cell lines in nude mice, and treatment with 100 microg of L2G7 or control given i.p. twice per week. RESULTS: MAb L2G7 strongly inhibited all biological activities of HGF measured in vitro, including cell proliferation, cell scattering, and endothelial tubule formation. Treatment with L2G7 completely inhibited the growth of established s.c. xenografts in nude mice. Moreover, systemic administration of L2G7 from day 5 induced the regression of intracranial U87 xenografts and dramatically prolonged the survival of tumor-bearing mice from a median of 39 to >90 days. L2G7 treatment of large intracranial tumors (average tumor size, 26.7 mm(3)) from day 18 induced substantial tumor regression (control group, 134.3 mm(3); L2G7 treated group, 11.7 mm(3)) by day 29 and again prolonged animal survival. CONCLUSIONS: These findings show that blocking the HGF-Met interaction with systemically given anti-HGF mAb can have profound antitumor effects even within the central nervous system, a site previously believed to be resistant to systemic antibody-based therapeutics.

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway.

In mammalian cells, the second messenger (2'-5',3'-5') cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP), is produced by the cytosolic DNA sensor cGAMP synthase (cGAS), and subsequently bound by the stimulator of interferon genes (STING) to trigger interferon response. Thus, the cGAS-cGAMP-STING pathway plays a critical role in pathogen detection, as well as pathophysiological conditions including cancer and autoimmune disorders. However, studying and targeting this immune signaling pathway has been challenging due to the absence of tools for high-throughput analysis. We have engineered an RNA-based fluorescent biosensor that responds to 2',3'-cGAMP. The resulting "mix-and-go" cGAS activity assay shows excellent statistical reliability as a high-throughput screening (HTS) assay and distinguishes between direct and indirect cGAS inhibitors. Furthermore, the biosensor enables quantitation of 2',3'-cGAMP in mammalian cell lysates. We envision this biosensor-based assay as a resource to study the cGAS-cGAMP-STING pathway in the context of infectious diseases, cancer immunotherapy, and autoimmune diseases.