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CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target–crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target–crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target–crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA–target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.

CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes. [ABSTRACT FROM AUTHOR]

The effects of zeatin, salicylic acid, and plant oligoadenylates on susceptibility of wheat seedlings to powdery mildew and the shape of dose-response curves for immunomodulation were studied. Salicylic acid and zeatin produced complex dose-responses with upregulation or downregulation of plant susceptibility. Under different experimental conditions, the dose-response curve for zeatin varied in shape from a curve with a maximum to a curve with a minimum of susceptibility to powdery mildew. More complicated curves with a resistance region framed with regions of increased susceptibility were also found. Plant oligoadenylates inhibited the development of powdery mildew at optimal concentrations. The increasing concentrations of salicylic acid and plant oligoadenylates, when combined with zeatin, led to a gradual change in the shape of the zeatin concentration curve. Treatment with salicylic acid and plant oligoadenylates in these experiments may simulate plant responses under biotic and abiotic stresses. Thus, exogenous zeatin was applied to model changes in the phytohormone metabolism. The complex multiphase nature of dose-response is proposed to cause a kind of random-number generator that produces variability in the physiological and immunological status of plants in natural conditions. Variation in susceptibility of individual plants may be crucial for the survival of infected plants and the stabilization of plant-pathogen interactions. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthetic development of modified nucleotides improves DMPK and pharmacodynamics. • Understanding cellular uptake mechanisms will improve targeted drug delivery. • Preclinical animal data scale well to humans, supporting dose-setting in clinical trials. • Improved bioanalytical sensitivity is needed to establish long elimination half-lives. • Mastering oral delivery will fuel interest from the industry and boost science. Oligonucleotide and modified mRNA therapeutics have great potential to treat diseases that are currently challenging to cure and are expanding into global and chronic disease areas such as cancer and various cardiovascular diseases. Advanced drug delivery systems or ligand–drug conjugates are utilized to achieve 'the right dose to the right target' to benefit efficacy and safety in patients. Chemistry and ADME characteristics distinguish these therapeutics from small molecules. Understanding the scalability and translatability between species and compound properties is crucial for robust nonclinical PKPD predictions to support clinical study design. Although the field has been developing for three decades, there is still room for innovation but also a need for nonclinical regulatory guidance to address these new modalities. Teaser Oligonucleotide-based therapeutics show great clinical promise and drug metabolism and pharmacokinetic understanding will be key to expand into broader patient populations. [ABSTRACT FROM AUTHOR]

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38β; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 μg/10 μl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38β MAPK ASO. Intrathecal injection of p38α or p38β ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38β mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38β MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions. [ABSTRACT FROM AUTHOR]

Context Toll-like receptor 4 (TLR4) activation contributes to obesity-associated insulin resistance in skeletal muscles (SM). TLR4 signaling involves two pathways: the myeloid differentiation primary response gene 88 (MyD88) leading to inflammatory cytokines production and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I (TRIF)-dependent pathways leading to type 1 interferon (IFNI) and interferon stimulated genes (ISG) expression. The E3 ubiquitin ligase RNF41 allows the preferential activation of the TRIF-IFNI pathway; however, its role in insulin response has not been reported. Methods We measured RNF41 level and IFNI pathway activation (ISG expression) in SM biopsies of obese insulin sensitive (OIS) and obese insulin resistant (OIR) women. Then we isolated and differentiated in myotubes, primary human SM cell progenitors from OIS and OIR SM biopsies. We modulated RNF41 and ISG expression in these myotubes and investigated their effects on insulin response. Results RNF41 expression is down-regulated in vivo in OIR SM and myotubes compared to OIS SM and myotubes. TLR4 activation with palmitate induces TRIF-IFNI pathway and ISG in OIS myotubes but not in OIR myotubes. Inhibition of RNF41 expression with siRNF41 in OIS myotubes treated with palmitate attenuates insulin response, IFNI pathway activation and ISG induction, mimicking OIR phenotype. Further, overexpression of RNF41 in OIR myotubes increases insulin response and ISG expression. Exposure to IFNI or to its inducer polyinosinic-polycytidylic acid, restores ISG expression and insulin sensitivity in OIR myotubes and OIS myotubes transfected with siRNF41. Conclusion Our results identify RNF41 as essential to IFNI pathway activation in order to maintain muscle insulin sensitivity during human obesity. [ABSTRACT FROM AUTHOR]

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-sea", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 X 10[sup 5] aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing. [ABSTRACT FROM AUTHOR]

Infections with viral pathogens impose considerable selective pressure on host defensive genes. Those genes at the forefront, responsible for identifying and binding exogenous molecular viral components, will carry the hallmarks of this struggle. Oligoadenylate synthetase (OAS) enzymes play a major role in the innate defense against a large number of viruses by acting as sensors of viral infections. Following their up-regulation by the interferon pathway, OASs bind viral dsRNA and then signal ribonuclease L (RNase L) to degrade RNA, shutting down viral and host protein synthesis. We have investigated the evolution of OAS1 in twenty-two Old World monkey species. We identified a total of 35 codons with the earmarks of positive selection and we performed a comprehensive analysis of their functional significance using in silico modeling of the OAS1 protein. Subdividing OAS1 into functional domains revealed intense purifying selection in the active domain but significant positive directional selection in the RNA-binding domain (RBD), the region where OAS1 binds viral dsRNA. The modeling analysis revealed a concentration of rapidly evolving residues in one region of the RBD suggestive of the sub-functionalization of different regions of the RBD. This analysis also identified several positively selected residues circumscribing the entry to the active site suggesting adaptive evasion of viral antagonism and/or selection for production of oligoadenylate of different length. [ABSTRACT FROM AUTHOR]

RNase L is activated by 2′,5′-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2′-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5′-modified 2-5A analogs with 8-methyladenosine at the 2′-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5′-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells. [ABSTRACT FROM AUTHOR]

The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2'-5' oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2'-5'-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2'-5'-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. [ABSTRACT FROM AUTHOR]

Interferons (IFN), first described 1957 by Isaacs and Lindemann, are antiviral proteins generated in cells after viral infections. One of several interferon-induced effector mechanisms is the so called 2-5A / RNaseL system: Interferon is produced in the virus-affected cells and released. After binding to cell membrane receptors of adjacent cells, 2-5 A synthetase (oligoadenylate synthetase, OAS) is generated, attaches to dsRNA section areas of the viral RNA and catalyses the production of 2-5 oligoadenylates from ATP. In 2-5 oligoadenylates, several adenosine residues (3-4 and more) are combined via phosphodiester binding in the unusual 2'-5' positions of the riboses. 2-5 oligoadenylates activate a RNaseL which degrades the viral RNA. Recently, characteristic gene mutations and other disturbances concerning the interferon system were detected in patients with severe COVID-19, leading to problems of 2-5 oligoadenylate synthesis and the activation of RNAseL. In order to circumvent these problems, we hypothesize that a direct application of 2-5 oligoadenylates, included in an inhalation spray, may be effective in treatment of severe COVID-19 infections of the respiratory system. In contrast to some other anti-COVID-19 drugs, oligoadenylates act inside the cells (like e.g. Paxlovid) and are therefore independent of cell surface mutations of the virus. For confirmation of our hypothesis, proof of concept investigations in vitro are suggested, before a possible clinical application can be considered. [ABSTRACT FROM AUTHOR]

Chromosomal aberrations in the product of conceptions (POC) are detected in around 50% lost pregnancies. Karyotype analysis of the POC, limited by high failure rate of culture, is not always carried out. The oligonucleotide-based chromosomal microarray analysis (CMA) of POC from 303 pregnancies was carried out. The findings showed a high diagnostic value of the test and its ability to detect a large number of clinically relevant rearrangements compared to karyotype analysis and comparative genomic hybridization (CGH) arrays. [ABSTRACT FROM PUBLISHER]

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination. [ABSTRACT FROM AUTHOR]

Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms. [ABSTRACT FROM AUTHOR]

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids. [ABSTRACT FROM AUTHOR]

[Display omitted] The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5′-phosphorylated 2′,5′-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5′-phosphate end on the activation of human RNase L, four sets of 5′-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5′-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC 50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity. [ABSTRACT FROM AUTHOR]

The article discusses study by Kazlauskiene et al. and Niewoehner et al in the issue about intracellular signalling in clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) immunity. Topics discussed include generation of cyclic-oligoadenylate (cOA), foreign DNA targeted by CRISPR-Cas types and RNase activity of Csm6 induced by cOA.

Summary Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5′ tag of crRNA, the 3′ anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation. Graphical Abstract Highlights • Crystal structure of the type III-A CRISPR-RNA-guided surveillance complex • Cryo-EM structures of cognate and non-cognate target RNA-bound Csm complexes • Complementary and non-complementary 3′ anti-tag binds at two distinct sites • Target RNA binding allosterically activates ssDNA cleavage and cOA synthesis Structures of the type III-A CRISPR-Csm complex in the presence of target RNAs reveal the molecular basis for a unique mode of self/non-self discrimination. [ABSTRACT FROM AUTHOR]

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric αβγ complex that functions as a central regulator of energy homeostasis. Energy stress manifests as a drop in the ratio of adenosine triphosphate (ATP) to AMP/ADP, which activates AMPK's kinase activity, allowing it to upregulate ATP-generating catabolic pathways and to reduce energy-consuming catabolic pathways and cellular programs. AMPK senses the cellular energy state by competitive binding of the three adenine nucleotides AMP, ADP, and ATP to three sites in its γ subunit, each, which in turn modulates the activity of AMPK's kinase domain in its α subunit. Our current understanding of adenine nucleotide binding and the mechanisms by which differential adenine nucleotide occupancies activate or inhibit AMPK activity has been largely informed by crystal structures of AMPK in different activity states. Here we provide an overview of AMPK structures, and how these structures, in combination with biochemical, biophysical, and mutational analyses provide insights into the mechanisms of adenine nucleotide binding and AMPK activity modulation. [ABSTRACT FROM AUTHOR]

The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction. [ABSTRACT FROM AUTHOR]