Recognition Of Nucleic Acids Research Articles

Sequence determinants in the cathelicidin LL-37 that promote inflammation via presentation of RNA to scavenger receptors

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure–function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term “innate immune vetting” to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.

Multisystem inflammation and susceptibility to viral infections in human ZNFX1 deficiency

Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.

Synthesis, Self‐Assembly, and Nucleic Acid Recognition of an Acylhydrazone‐Conjugated Cationic Tetraphenylethene Ligand

AbstractSupramolecular polymers are of interest in the pursuit of multivalent nucleic acids recognition. However, their formation often relies on non‐covalent forces that are also at play in the interaction with nucleic acids. In this work, we designed a novel compound (TPE‐Gir) combining a tetraphenylethene aromatic core tethered to four quaternary ammoniums through acylhydrazone spacers, and we investigated in detail its self‐assembly and interaction with different types of nucleic acids. The spectroscopic analyses indicate the self‐assembly of regular fluorescent nanoparticles (observed by DLS and TEM) in the absence of nucleic acids, the strong propensity to intercalate into single‐stranded DNA, the ability to bind into the minor groove of double‐stranded DNA, and the selective binding to G‐quadruplex (G4) structures by fitting within a wide G4‐groove. Those recognition events are quantified by isothermal titration calorimetry and the proposed binding models are supported by docking simulations.

How Enzymes, Proteins, and Antibodies Recognize Extended DNAs; General Regularities.

X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their total affinity for enzymes and proteins. The interaction of different enzymes and proteins with long DNA and RNA at the quantitative molecular level can be successfully analyzed using the method of the stepwise increase in ligand complexity (SILC). The present review summarizes the data on stepwise increase in ligand complexity (SILC) analysis of nucleic acid recognition by various enzymes—replication, restriction, integration, topoisomerization, six different repair enzymes (uracil DNA glycosylase, Fpg protein from Escherichia coli, human 8-oxoguanine-DNA glycosylase, human apurinic/apyrimidinic endonuclease, RecA protein, and DNA-ligase), and five DNA-recognizing proteins (RNA helicase, human lactoferrin, alfa-lactalbumin, human blood albumin, and IgGs against DNA). The relative contributions of structural elements of DNA fragments “covered” by globules of enzymes and proteins to the total affinity of DNA have been evaluated. Thermodynamic and catalytic factors providing discrimination of unspecific and specific DNAs by these enzymes on the stages of primary complex formation following changes in enzymes and DNAs or RNAs conformations and direct processing of the catalysis of the reactions were found. General regularities of recognition of nucleic acid by DNA-dependent enzymes, proteins, and antibodies were established.

Signaling Through Nucleic Acid Sensors and Their Roles in Inflammatory Diseases.

Recognition of pathogen-derived nucleic acids by pattern-recognition receptors (PRRs) is essential for eliciting antiviral immune responses by inducing the production of type I interferons (IFNs) and proinflammatory cytokines. Such responses are a prerequisite for mounting innate and pathogen-specific adaptive immune responses. However, host cells also use nucleic acids as carriers of genetic information, and the aberrant recognition of self-nucleic acids by PRRs is associated with the onset of autoimmune or autoinflammatory diseases. In this review, we describe the mechanisms of nucleic acid sensing by PRRs, including Toll-like receptors, RIG-I-like receptors, and DNA sensor molecules, and their signaling pathways as well as the disorders caused by uncontrolled or unnecessary activation of these PRRs.

Divergent effects of acute versus chronic glucocorticoids in COVID-19.

Divergent effects of acute versus chronic glucocorticoids in COVID-19.

Structural basis for multifunctional roles of human Ints3 C-terminal domain

Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1), and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

Structure of the miniature type V-F CRISPR-Cas effector enzyme

RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Nobel Prize in Chemistry 2020: The great breakthrough from frontier research in microbiology

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for their joint development of the highly efficient CRISPR-Cas9 genome editing tools, which have taken the life sciences into a new era. The CRISPR-Cas system was first discovered in bacteria in 1987. After more than 20 years of arduous exploration of these enigmatic systems that were found in more than 90% archaea and about half bacteria, their antiviral function was finally confirmed in 2007. This has inspired more researchers to focus on the CRISPR-Cas systems, of which the general anti-virus mechanisms were also uncovered within the next few years, that the crRNA containing the virus sequence information guides the Cas protein(s) to recognize the corresponding virus and cleave their DNA or RNA. Among those pioneers in this field, Charpentier and Doudna mainly focused on the type II CRISPR-Cas9 system, which is much simpler than the wide-spread type I systems. In 2012, Doudna and Charpentier had elucidated the fine structure of the anti-virus effector complex of the CRISPR-Cas9 system, which contains a single Cas9 protein and two RNA components (crRNA and tracrRNA). It was this work that led to the breakthrough in developing the CRISPR-Cas9 genome editing tools. Significantly, they innovatively connected the tracrRNA and crRNA into a single RNA chimera (sgRNA), greatly simplifying the genome editing tools to only two components, the sgRNA and the single Cas9 protein. Compared with the genome editing tools developed in the past (Meganuclease, ZFN and TALEN), CRISPR-Cas9 has significant advantages such as easy programming, high efficiency and a wide application range. These merits make CRISPR-Cas9 so far the most widely used genome editing technique. In basic research, CRISPR-Cas9 genome editing has been used to elucidate the functions of unknown genes or pathways. In application, this technique has greatly improved the efficiency of animal and plant breeding, and made it possible to cure some genetic diseases and even cancers. Moreover, based on this CRISPR-Cas9 platform, other advanced genome editing tools have also been developed, such as base editing and prime editing, etc. It is also believed that, in addition to CRISPR-Cas systems, there are still many other undiscovered antiviral systems in microbes, as well as a large number of unknown nucleic acid recognition and processing elements, which are expected to optimize or upgrade the current genome editing tools, or even innovate entirely new genome editing tools. The CRISPR-Cas genome editing tools were developed from the bacterial anti-virus immune systems, thus the 2020 Nobel Prize in Chemistry is essentially a reward to the frontier research in microbiology. In this article, we will briefly introduce i) the timeline for the development of genome editing tools; ii) the historical background of the CRISPR-Cas research; iii) the breakthrough works for the birth of CRISPR-Cas9 genome editing technology. The future frontier research in microbiology and big data mining to further optimize or innovate genome editing tools is also prospected.

Three-way junction DNA based electrochemical biosensor for microRNAs detection with distinguishable locked nucleic acid recognition and redox cycling signal amplification

Herein, we present one three-way junction (TWJ) DNA based electrochemical biosensor for sensitive microRNAs detection with locked nucleic acid (LNA) as capture probe and enzyme-free redox cycling signal amplification strategy. LNA owns higher binding affinity and thermal stability compared to normal DNA sequence. In the presence of target microRNA (miRNA-21), novel TWJ structure can be specially triggered with the assistant of methylene blue (MB)-tagged DNA signal probe, thus producing an enhanced electrochemical current. Furthermore, a chemical reductant, tris (2-carboxyethyl) phosphine (TCEP), is adopted to lead a relayed electron transport between solution TCEP and TWJ-linked MB tag and amplified whole signal intensity. Based on the selectivity from target-induced LNA/DNA TWJ structure and enzyme-free MB cycling amplification strategy, the designed electrochemical biosensor exhibits sensitive miRNA-21 detection, with a linear range from 100 aM to 100 pM and a low detection limit of 77 aM. In addition, it can successfully discriminate the differences between the miRNA family members, presenting a promising response to the direct determination of target miRNA-21 in real biological fluids. Our work provides an alternative LNA-based electrochemical biosensor way in the area of miRNA diagnostics and clinical analysis without complicated enzyme/nanomaterials-assisted signal amplification.

Analysis of Sars-Cov-2-Associated Proteins Identify Tank-Binding Kinase-1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency. Importantly, recent studies highlighted the severity of COVID-19 in MM. To date, however, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. Plasmacytoid dendritic cells (pDCs) play a key role in both recognition of viral nucleic acid and initiating anti-viral activity via type 1 interferon (IFN) response signaling. We showed that interactions of dysfunctional pDCs with MM cells, T cells, and NK cells confer immune suppression in the host-MM bone marrow (BM) microenvironment (Chauhan et al, Cancer Cell 2009; 16: p309; Ray et al, Leukemia 2015; 29: p1441). Here, we analyzed the immune-pathway proteins implicated in Covid-19-host interactions [Gordon et al, Nature 2020; 583, p459] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. We identified the TLR pathway serine/threonine kinase TBK1 (TANK-binding kinase 1), involved in type I IFN induction, as a potential immunotherapeutic target in MM. Moreover, Covid-19 relies on host-Ubiquitin-proteasome-system for propagation, and we found that targeting ubiquitin receptor Rpn13 with a specific inhibitor RA190 restores TBK1 expression in MM.Methods For our studies, we examined SARS-CoV-2-human protein-protein interactions (PPIs) maps (Gordon et al, 2020). pDC-MM co-cultures, and RNAseq using next- generation sequencing (NGS): Purified MM patient pDCs were cocultured with autologous MM cells (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using FACS. Total RNA from MM cells was subjected to RNAseq analysis using Illumina NGS. Raw sequence data were analyzed to generate differential expression (DEseq2). Linear model (Limma) and its GUI (Glimma) were also used. Statistical significance: log2FC (fold change) values in coculture vs control, with a False Discovery Rate value of <0.05, was considered significant (CI > 95). The heatmap analysis was done using Morpheus software (Broad Institute, MIT). MM patient and pDCs data used for bioinformatics were from the NCBI GEO.Results A total of 41 genes involved in the Covid-19 host-pathogen immune interactions are also differentially expressed in MM after coculture with pDCs. (log2Fold change: ± 3.5-fold; p < 0.00001; pDC-MM vs MM alone). The gene expression widely varies in pDC-MM vs MM [Median (log10): -0.13 to 4.5; adj p < 0.001]. We identified 3 specific innate immunity-linked genes TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor) which are essential for IFN production in MM. Importantly, pDCs decrease TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) as well as induce IRF3 (Log2FC: 1.5) in MM (p < 0.0001). Analysis of publicly available gene profiling datasets on relapsed MM patient showed low levels of TBK1 and RAE1 and higher IRF3 (n = 50) [Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, low TBK1 expression correlates with poor survival in MM patients (n = 350) and elevated TBK1 correlates with a better prognosis (p = 0.026). Similar analysis showed that most of 41 genes involved in the Covid-19 host-pathogen immune interactions are expressed in pDCs. In unstimulated pDCs, TBK1 expression is significantly lower than IRF3 and RAE1 (2-3-fold, p < 0.05). In functional studies, treatment of pDCs with IFN-α activating CpG-ODN type-A increases both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043), without significantly altering IRF3 expression. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs-MM interactions further decreases TBK1 in MM, thereby attenuating IFN-mediated anti-viral immune response signaling in MM. Finally, we found that blockade of proteasome-mediated protein degradation via inhibition of Ubiquitin receptor Rpn13 upregulates TBK-1 expression, indicating potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM.Conclusion We found that pDC-MM interactions downregulate TBK1, which in turn reduces IFN response signaling essential to generate an effective anti-viral immune response in the MM BM microenvironment. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19; and 2) targeting TBK1 may restore anti-viral immune response and reduce COVID-19 severity in MM.DisclosuresChauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

MAVS regulates the quality of the antibody response to West-Nile Virus.

A key difference that distinguishes viral infections from protein immunizations is the recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs). Insights into the functions of cytosolic PRRs such as the RNA-sensing Rig-I-like receptors (RLRs) in the instruction of adaptive immunity are therefore critical to understand protective immunity to infections. West Nile virus (WNV) infection of mice deficent of RLR-signaling adaptor MAVS results in a defective adaptive immune response. While this finding suggests a role for RLRs in the instruction of adaptive immunity to WNV, it is difficult to interpret due to the high WNV viremia, associated exessive antigen loads, and pathology in the absence of a MAVS-dependent innate immune response. To overcome these limitations, we have infected MAVS-deficient (MAVSKO) mice with a single-round-of-infection mutant of West Nile virus. We show that MAVSKO mice failed to produce an effective neutralizing antibody response to WNV despite normal antibody titers against the viral WNV-E protein. This defect occurred independently of antigen loads or overt pathology. The specificity of the antibody response in infected MAVSKO mice remained unchanged and was still dominated by antibodies that bound the neutralizing lateral ridge (LR) epitope in the DIII domain of WNV-E. Instead, MAVSKO mice produced IgM antibodies, the dominant isotype controlling primary WNV infection, with lower affinity for the DIII domain. Our findings suggest that RLR-dependent signals are important for the quality of the humoral immune response to WNV.

The immunorecognition, subcellular compartmentalization, and physicochemical properties of nucleic acid nanoparticles can be controlled by composition modification.

Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2′F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2′F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.

Intracellular PRRs Activation in Targeting the Immune Response Against Fungal Infections.

The immune response against fungal infections is complex and exhibits several factors involving innate elements that participate in the interaction with the fungus. The innate immune system developed pattern recognition receptors that recognize different pathogen-associated molecular patterns present both on the surface of the fungi cell wall and on their genetic material. These receptors have the function of activating the innate immune response and regulating a subsequent adaptive immune response. Among pattern recognition receptors, the family of Toll-like receptors and C-type lectin receptors are the best described and characterized, they act directly in the recognition of pathogen-associated molecular patterns expressed on the wall of the fungus and consequently in directing the immune response. In recent years, the role of intracellular pattern recognition receptors (TLR3, TLR7, TLR8, and TLR9) has become increasingly important in the pathophysiology of some mycoses, as paracoccidioidomycosis, cryptococcosis, aspergillosis, and candidiasis. The recognition of nucleic acids performed by these receptors can be essential for the control of some fungal infections, as they can be harmful to others. Therefore, this review focuses on highlighting the role played by intracellular pattern recognition receptors both in controlling the infection and in the host's susceptibility against the main fungi of medical relevance.

Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish.

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.

Unnatural bases for recognition of noncoding nucleic acid interfaces.

The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co-opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.

The Ubiquitin E3 Ligase Parkin Inhibits Innate Antiviral Immunity Through K48-Linked Polyubiquitination of RIG-I and MDA5.

Innate immunity is the first-line defense against antiviral or antimicrobial infection. RIG-I and MDA5, which mediate the recognition of pathogen-derived nucleic acids, are essential for production of type I interferons (IFN). Here, we identified mitochondrion depolarization inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibited the response and antiviral activity of type I IFN during viral infection. Furthermore, we found that the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase Parkin mediated mitophagy, thus negatively regulating the activation of RIG-I and MDA5. Parkin directly interacted with and catalyzed the K48-linked polyubiquitination and subsequent degradation of RIG-I and MDA5. Thus, we demonstrate that Parkin limits RLR-triggered innate immunity activation, suggesting Parkin as a potential therapeutic target for the control of viral infection.

DNA targeting and interference by a bacterial Argonaute nuclease.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.

Protein Spherical Nucleic Acids for Live-Cell Chemical Analysis.

We report the development of a new strategy for the chemical analysis of live cells based on protein spherical nucleic acids (ProSNAs). The ProSNA architecture enables analyte detection via the highly programmable nucleic acid shell or a functional protein core. As a proof-of-concept, we use an i-motif as the nucleic acid recognition element to probe pH in living cells. By interfacing the i-motif with a forced-intercalation readout, we introduce a quencher-free approach that is resistant to false-positive signals, overcoming limitations associated with conventional fluorophore/quencher-based gold NanoFlares. Using glucose oxidase as a functional protein core, we show activity-based, amplified sensing of glucose. This enzymatic system affords greater than 100-fold fluorescence turn on in buffer, is selective for glucose in the presence of close analogs (i.e., glucose-6-phosphate), and can detect glucose above a threshold concentration of ∼5 μM, which enables the study of relative changes in intracellular glucose concentrations.