Nucleic Acid Binding Research Articles

Abstract P3-09-07: R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers

Abstract Transcriptional R-loops are triple-stranded RNA:DNA hybrid genome structures which are defined in more than 75% of genes of the human genome, functionally versatile in cells, and mechanistically involved in tumorigenesis. In cancer cells, excessive/aberrant R-loop structures promote mutagenesis and genome instability due to their key regulatory roles in transcription and genotoxic stress. Thus, a balance in the regulation of R-loop initiation, stabilization, and suppression is required for proper function. Recent studies have shown that R-loop formation increases in luminal but not in basal-like cells of the tumor-free mammary epithelium of BRCA1 mutation carriers. R-loop forming sequences (RLFSs) are strand-specific G-rich regions of ssDNA that initiate and stabilize the formation of R-loops. The roles of the RLFSs in functional R-loops associated with premalignant states of BRCA1-mc have not been studied. Using our QmRLFS model and R-loopDB (http://rloop.bii.a-star.edu.sg/), we identified RLFS-associated (RLFS(+)) and non-associated (RLFS(-)) RNA:DNA hybrids detected by published DRIP-seq data in gene regions in luminal precursors, mature luminal, and basal-like epithelial cell type populations, extracted from BRCA1 wild-type and mutated non-cancer breast tissue samples (GSE96672). We found that genome regions of RLFS(+) RNA:DNA hybrid signals are strongly associated with G4-quadruplex, ssDNA regions, histone markers (acetylation, demethylation), and other open chromatin markers while the RLFS(-) RNA:DNA hybrid signal regions were not significantly associated with the positions of these regulatory markers. Our statistical modeling and cell-type-specific gene analysis of normal and BRCA1-deficient non-cancer epithelial cells samples shown that RLFS(+) R-loop frequency in luminal cells was mostly increased; in contrast, RLFS(+) R-loops were essentially decreased in basal-like breast cells. Analyzing published gene expression profiles of mammary epithelial cells obtained from disease-free prophylactic mastectomy tissues of BRCA1-mutation carriers and reduction mammoplasty tissues from non-mutation carriers (GSE25835 & GSE19383), we found the differential expressed genes (DEGs) associated with RLFS(+) RNA-DNA hybrid (DRIP-seq) signals. Integrating these findings with our RLFS RNA-DNA hybrid characterizations, we dichotomized the DEGs referring to the luminal and basal-like cell types. According to pathway analysis, a majority of BRCA1-deficient altered transcripts referring to luminal cell’s genes are involved metabolism and cell cycle while basal-like cells involved immune functions, cell signaling, and epithelial-mesenchymal transition pathways. We also found that the genes associated with RLFS clusters present on the sense strand are more associated with nucleic acid binding and transcription regulation, while the genes with an RLFS present on the anti-sense strand are more associated with immune system functions depleted in BRCA1 deficient non-cancer carriers. Thus, for the first time, we demonstrate the roles of RLFS mediated R-looping in the luminal and basal-like breast cells from BRCA1-mutation carrier tissues that specify aberrantly transcribed genes involving in tumorigenesis and immune function pathways. This study suggests novel regulatory mechanics of BRCA1-driven pre-malignant state development and prospective biomarker discovery. Citation Format: Andre Grageda, Vladimir A Kuznetsov. R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-07.

Assessment of photoacoustic tomography contrast for breast tissue imaging using 3D correlative virtual histology

Current breast tumor margin detection methods are destructive, time-consuming, and result in significant reoperative rates. Dual-modality photoacoustic tomography (PAT) and ultrasound has the potential to enhance breast margin characterization by providing clinically relevant compositional information with high sensitivity and tissue penetration. However, quantitative methods that rigorously compare volumetric PAT and ultrasound images with gold-standard histology are lacking, thus limiting clinical validation and translation. Here, we present a quantitative multimodality workflow that uses inverted Selective Plane Illumination Microscopy (iSPIM) to facilitate image co-registration between volumetric PAT-ultrasound datasets with histology in human invasive ductal carcinoma breast tissue samples. Our ultrasound-PAT system consisted of a tunable Nd:YAG laser coupled with a 40 MHz central frequency ultrasound transducer. A linear stepper motor was used to acquire volumetric PAT and ultrasound breast biopsy datasets using 1100 nm light to identify hemoglobin-rich regions and 1210 nm light to identify lipid-rich regions. Our iSPIM system used 488 nm and 647 nm laser excitation combined with Eosin and DRAQ5, a cell-permeant nucleic acid binding dye, to produce high-resolution volumetric datasets comparable to histology. Image thresholding was applied to PAT and iSPIM images to extract, quantify, and topologically visualize breast biopsy lipid, stroma, hemoglobin, and nuclei distribution. Our lipid-weighted PAT and iSPIM images suggest that low lipid regions strongly correlate with malignant breast tissue. Hemoglobin-weighted PAT images, however, correlated poorly with cancerous regions determined by histology and interpreted by a board-certified pathologist. Nuclei-weighted iSPIM images revealed similar cellular content in cancerous and non-cancerous tissues, suggesting malignant cell migration from the breast ducts to the surrounding tissues. We demonstrate the utility of our nondestructive, volumetric, region-based quantitative method for comprehensive validation of 3D tomographic imaging methods suitable for bedside tumor margin detection.

Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases.

Biomedical researchers have subsequently been inspired the development of new approaches for precisely changing an organism’s genomic DNA in order to investigate customized diagnostics and therapeutics utilizing genetic engineering techniques. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one such technique that has emerged as a safe, targeted, and effective pharmaceutical treatment against a wide range of disease-causing organisms, including bacteria, fungi, parasites, and viruses, as well as genetic abnormalities. The recent discovery of very flexible engineered nucleic acid binding proteins has changed the scientific area of genome editing in a revolutionary way. Since current genetic engineering technique relies on viral vectors, issues about immunogenicity, insertional oncogenesis, retention, and targeted delivery remain unanswered. The use of nanotechnology has the potential to improve the safety and efficacy of CRISPR/Cas9 component distribution by employing tailored polymeric nanoparticles. The combination of two (CRISPR/Cas9 and nanotechnology) offers the potential to open new therapeutic paths. Considering the benefits, demand, and constraints, the goal of this research is to acquire more about the biology of CRISPR technology, as well as aspects of selective and effective diagnostics and therapies for infectious illnesses and other metabolic disorders. This review advocated combining nanomedicine (nanomedicine) with a CRISPR/Cas enabled sensing system to perform early-stage diagnostics and selective therapy of specific infectious disorders. Such a Nano-CRISPR-powered nanomedicine and sensing system would allow for successful infectious illness control, even on a personal level. This comprehensive study also discusses the current obstacles and potential of the predicted technology.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s40097-022-00472-7.

Insight into SAMHD1 regulation mechanism using molecular dynamics simulations

SAMHD1 is a dNTP triphosphohydrolase that is implicated in retroviral restriction in myeloid cells. Since SAMHD1 regulates dNTP pools it also has important role in cellular homeostasis, cell cycle regulation, and innate immunity. The exact mechanism of the retroviral restriction by SAMHD1 is not yet known as contradictory results between dNTPase and antiretroviral activity was observed where loss in antiretroviral activity did not correlate with its retention of dNTPase activity. Regulation of SAMHD1 activity may be a complex interplay of redox action, phosphorylation and nucleic acid binding.

Binding and compaction function of structural domains of SARS-CoV-2 nucleocapsid (N) protein on a single stranded nucleic acid substrate

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes for four major structural proteins. The nucleocapsid (N) protein is highly conserved among coronaviruses and is the only structural protein that binds the viral RNA packaged in the virion. One function of the N protein is the formation of a helical ribonucleoprotein (RNP) structure, which organizes and compacts the viral RNA inside the virion This process requires both nucleic-acid (NA) binding and interprotein interactions, which have been associated with the two structured N-terminal and C-terminal domains (NTD and CTD) within the N protein.

Characterizing nucleic acid interactions with the N-Terminal binding domain of the SARS-CoV-2 nucleocapsid protein

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Quantifying the mechanisms controlling nucleic acid binding is a key step to understand how such condensation is realized. We have previously shown that the three disordered regions flanking the RNA binding domain (RBD) and dimerization domain are highly dynamic. Here, we focus on the role of the RBD and the disordered N-Terminal Domain (NTD) tail. Using single-molecule Foerster Resonance Energy Transfer, we quantify both binding of the nucleic acids and concomitant conformational changes occurring in the disordered region.

Competing interactions regulate the granularity of the nucleolus

Ribosome biogenesis is a critical cellular process and its dysregulation is associated with morbidity and death. Biogenesis and assembly of these complex molecular machines occurs in the nucleolus, a large, multi-compartment membrane-less organelle formed through liquid-liquid phase separation (LLPS). The protein Nucleophosmin (NPM1) is a major constituent of the outer, granular component (GC) region of the nucleolus where it phase separates with ribosomal proteins (r-proteins) and nucleolar proteins containing disordered Arginine-rich regions using its central intrinsically disordered region, and with ribosomal RNAs (rRNA) using its folded C-terminal nucleic acid binding domain.

DAMPs/PAMPs induce monocytic TLR activation and tolerance in COVID-19 patients; nucleic acid binding scavengers can counteract such TLR agonists

Millions of COVID-19 patients have succumbed to respiratory and systemic inflammation. Hyperstimulation of toll-like receptor (TLR) signaling is a key driver of immunopathology following infection by viruses. We found that severely ill COVID-19 patients in the Intensive Care Unit (ICU) display hallmarks of such hyper-stimulation with abundant agonists of nucleic acid-sensing TLRs present in their blood and lungs. These nucleic acid-containing Damage and Pathogen Associated Molecular Patterns (DAMPs/PAMPs) can be depleted using nucleic acid-binding microfibers to limit the patient samples’ ability to hyperactivate such innate immune receptors. Single-cell RNA-sequencing revealed that CD16+ monocytes from deceased but not recovered ICU patients exhibit a TLR-tolerant phenotype and a deficient anti-viral response after ex vivo TLR stimulation. Plasma proteomics confirmed such myeloid hyperactivation and revealed DAMP/PAMP carrier consumption in deceased patients. Treatment of these COVID-19 patient samples with MnO nanoparticles effectively neutralizes TLR activation by the abundant nucleic acid-containing DAMPs/PAMPs present in their lungs and blood. Finally, MnO nanoscavenger treatment limits the ability of DAMPs/PAMPs to induce TLR tolerance in monocytes. Thus, treatment with microfiber- or nanoparticle-based DAMP/PAMP scavengers may prove useful for limiting SARS-CoV-2 induced hyperinflammation, preventing monocytic TLR tolerance, and improving outcomes in severely ill COVID-19 patients.

Recent Advances in the Role of Arid5a in Immune Diseases and Cancer.

AT-rich interactive domain 5a (Arid5a) is a nucleic acid binding protein. In this review, we highlight recent advances in the association of Arid5a with inflammation and human diseases. Arid5a is known as a protein that performs dual functions. In in vitro and in vivo studies, it was found that an inflammation-dependent increase in Arid5a expression mediates both transcriptional and post-transcriptional regulatory effects that are implicated in immune regulation and cellular homeostasis. A series of publications demonstrated that inhibiting Arid5a augmented several processes, such as preventing septic shock, experimental autoimmune encephalomyelitis, acute lung injury, invasion and metastasis, immune evasion, epithelial-to-mesenchymal transition, and the M1-like tumor-associated macrophage (TAM) to M2-like TAM transition. In addition, Arid5a controls adipogenesis and obesity in mice to maintain metabolic homeostasis. Taken together, recent progress indicates that Arid5a exhibits multifaceted, both beneficial and detrimental, roles in health and disease and suggest the relevance of Arid5a as a potential therapeutic target.

Identification and Characterization of a Garlic Virus E Genome in Garlic (Allium sativum L.) Using High-Throughput Sequencing from India.

Garlic (Allium sativum L.) plants exhibiting mosaics, deformation, and yellow stripes symptoms were identified in Meerut City, Uttar Pradesh, India. To investigate the viruses in the garlic samples, the method of high-throughput sequencing (HTS) was used. Complete genome of the garlic virus E (GarV-E) isolate (NCBI accession No. MW925710) was retrieved. The virus complete genome comprises 8450 nucleotides (nts), excluding the poly (A) tail at the 3′ terminus, with 5′ and 3′ untranslated regions (UTRs) of 99 and 384 nts, respectively, and ORFs encoding replicase with a conserved motif for RNA-dependent RNA polymerase (RdRP), TGB1, TGB2, TGB3, serine-rich protein, coat protein, and nucleic acid binding protein (NABP). The sequence homology shared 83.49–90.40% and 87.48–92.87% with those of GarV-E isolates available in NCBI at the nucleotide and amino acid levels, respectively. Phylogenetic analysis showed a close relationship of this isolate from India (MW925710) with GarV-E isolate YH (AJ292230) from Zhejiang, China. The presence of GarV-E was also confirmed by RT-PCR. The present study is the first report of GarV-E in garlic cultivar Yamuna Safed-3 grown in northern India. However, further studies are needed to confirm its role in symptom development, nationwide distribution, genetic diversity, and potential yield loss to the garlic in India.

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

Monitoring functional RNA binding of RNA-dependent ATPase enzymes such as SF2 helicases using RNA dependent ATPase assays: A RIG-I case study.

The highly conserved Superfamily 1 (SF1) and Superfamily 2 (SF2) nucleic acid-dependent ATPases, are ubiquitous motor proteins with central roles in DNA and RNA metabolism (Jankowsky & Fairman, 2007). These enzymes require RNA or DNA binding to stimulate ATPase activity, and the conformational changes that result from this coupled behavior are linked to a multitude of processes that range from nucleic acid unwinding to the flipping of macromolecular switches (Pyle, 2008, 2011). Knowledge about the relative affinity of nucleic acid ligands is crucial for deducing mechanism and understanding biological function of these enzymes. Because enzymatic ATPase activity is directly coupled to RNA binding in these proteins, one can utilize their ATPase activity as a simple reporter system for monitoring functional binding of RNA or DNA to an SF1 or SF2 enzyme. In this way, one can rapidly assess the relative impact of mutations in the protein or the nucleic acid and obtain parameters that are useful for setting up more quantitative direct binding assays. Here, we describe a routine method for employing NADH-coupled enzymatic ATPase activity to obtain kinetic parameters reflecting apparent ATP and RNA binding to an SF2 helicase. First, we provide a protocol for calibrating an NADH-couple ATPase assay using the well-characterized ATPase enzyme hexokinase, which a simple ATPase enzyme that is not coupled with nucleic acid binding. We then provide a protocol for obtaining kinetic parameters (KmATP, Vmax and KmRNA) for an RNA-coupled ATPase enzyme, using the double-stranded RNA binding protein RIG-I as a case-study. These approaches are designed to provide investigators with a simple, rapid method for monitoring apparent RNA association with SF2 or SF1 helicases.

Binding Analysis Using Accelerated Molecular Dynamics Simulations and Future Perspectives.

Biomolecular recognition such as binding of small molecules, nucleic acids, peptides and proteins to their target receptors plays key roles in cellular function and has been targeted for therapeutic drug design. Molecular dynamics (MD) is a computational approach to analyze these binding processes at an atomistic level, which provides valuable understandings of the mechanisms of biomolecular recognition. However, the rather slow biomolecular binding events often present challenges for conventional MD (cMD), due to limited simulation timescales (typically over hundreds of nanoseconds to tens of microseconds). In this regard, enhanced sampling methods, particularly accelerated MD (aMD), have proven useful to bridge the gap and enable all-atom simulations of biomolecular binding events. Here, we will review the recent method developments of Gaussian aMD (GaMD), ligand GaMD (LiGaMD) and peptide GaMD (Pep-GaMD), which have greatly expanded our capabilities to simulate biomolecular binding processes. Spontaneous binding of various biomolecules to their receptors has been successfully simulated by GaMD. Microsecond LiGaMD and Pep-GaMD simulations have captured repetitive binding and dissociation of small-molecule ligands and highly flexible peptides, and thus enabled ligand/peptide binding thermodynamics and kinetics calculations. We will also present relevant application studies in simulations of important drug targets and future perspectives for rational computer-aided drug design.

Novel approaches to study helicases using magnetic tweezers.

Helicases form a universal family of molecular motors that bind and translocate onto nucleic acids. They are involved in essentially every aspect of nucleic acid metabolism: from DNA replication to RNA decay, and thus ensure a large spectrum of functions in the cell, making their study essential. The development of micromanipulation techniques such as magnetic tweezers for the mechanistic study of these enzymes has provided new insights into their behavior and their regulation that were previously unrevealed by bulk assays. These experiments allowed very precise measures of their translocation speed, processivity and polarity. Here, we detail our newest technological advances in magnetic tweezers protocols for high-quality measurements and we describe the new procedures we developed to get a more profound understanding of helicase dynamics, such as their translocation in a force independent manner, their nucleic acid binding kinetics and their interaction with roadblocks.

Histone H3 deacetylation promotes host cell viability for efficient infection by Listeria monocytogenes.

For many intracellular bacterial pathogens manipulating host cell survival is essential for maintaining their replicative niche, and is a common strategy used to promote infection. The bacterial pathogen Listeria monocytogenes is well known to hijack host machinery for its own benefit, such as targeting the host histone H3 for modification by SIRT2. However, by what means this modification benefits infection, as well as the molecular players involved, were unknown. Here we show that SIRT2 activity supports Listeria intracellular survival by maintaining genome integrity and host cell viability. This protective effect is dependent on H3K18 deacetylation, which safeguards the host genome by counteracting infection-induced DNA damage. Mechanistically, infection causes SIRT2 to interact with the nucleic acid binding protein TDP-43 and localise to genomic R-loops, where H3K18 deacetylation occurs. This work highlights novel functions of TDP-43 and R-loops during bacterial infection and identifies the mechanism through which L. monocytogenes co-opts SIRT2 to allow efficient infection.

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified ‘β-clasp’ structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.

Reversible superhydrophobicity unyielding magnetic beads of flipping-triggered (SYMBOL) regulate the binding and unbinding of nucleic acids for ultra-sensitive detection

Present molecular biology procedures often include extraction and purification of nucleic acids (NAs) based on centrifuge- and column-based methods, which involve the usage of specialized apparatus and toxic chemicals. These restrict its usage in large scale high-throughput settings. Magnetic beads (MBs) may provide an ideal approach to extracting and purifying DNA or RNA instead of them. However, the low separation rate of NAs (DNA or RNA) on MBs limits its NAs’ detection. Here, we developed a reversible superhydrophobicity unyielding magnetic-beads of flipping-triggered (SYMBOL) system which can regulate the binding and unbinding capacity of NAs via the buffers due to the flipping of –COO- on the surface of SYMBOL. Compared to other MBs examined in this study, SYMBOL exhibited similar binding properties and far enhanced separation properties. In fact, the DNA separation rate rose from about 79% to nearly 96%, whereas, the RNA separation rate increased from roughly 75% to 97%. When it applied to SARS-CoV-2 detection, the SYMBOL, coupled with Simulation Digital RT-LAMP (a previously developed system), could further increase the sensitivity of the detection, thereby achieving a detection threshold of 5 copies/mL. The SYMBOL showed excellent extraction capability in a variety of tissues and can be used in a wide range of nucleic acid related technologies such as PCR, sequencing, etc.

Abstract P135: Systemic targeting of a CNS tumor (medulloblastoma) using a novel cell-penetrating, nucleic acid binding, monoclonal antibody

Abstract Targeting tumors of the central nervous system remains challenging given the physiological barriers preventing the delivery of therapeutics across the blood brain barrier (BBB). This is particularly true for nucleic acids, which degrade rapidly in serum and often fail to penetrate cells even with proper formulation into AAVs or LNPs. To overcome these barriers, many current efforts rely on invasive methods of delivery, including intrathecal or intraparenchymal injection. Here we report studies evaluating the ability of a novel cell-penetrating antibody, GMAB, to penetrate the BBB and target a CNS model of medulloblastoma (DAOY) following intravenous administration. Studies with fluorescently labeled GMAB demonstrated that the antibody readily penetrated into the CNS following systemic administration, achieving tumor-specific delivery while sparing surrounding normal brain tissue. Subsequent studies with 3p-hpRNA, a known activator of RIG-I, a cytosolic pattern recognition receptor which detects viral RNA and elicits a type-I interferon response, demonstrated that GMAB could deliver RNA ligands to tumors, resulting in significantly reduced intracranial tumor burden 10 days after a single-dose administered systemically while also preventing the development of spinal metastases. These results highlight a novel approach for the systemic delivery of immunostimulatory RNAs in a targeted manner to multiple tumors including often difficult to treat CNS tumors, which may offer treatment advantages over current approaches. Citation Format: Elias Quijano, Minsoo Khang, Bruce C. Turner, Stephen Squinto, Ranjit Bindra, W. Mark Saltzman, Peter Glazer. Systemic targeting of a CNS tumor (medulloblastoma) using a novel cell-penetrating, nucleic acid binding, monoclonal antibody [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P135.

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, −2 and −3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.

Structural, energetic and lipophilic analysis of SARS-CoV-2 non-structural protein 9 (NSP9)

In SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19.