Nucleic Acid Processing Research Articles

DMF-Bimol: Counting mRNA and Protein Molecules in Single Cells with Digital Microfluidics.

Analyzing single-cell protein and mRNA levels yields invaluable insights into cellular functions and the intricacies of biologically heterogeneous systems. Current joint mRNAs and protein analysis methodologies suffer from relative quantification, low sensitivity, possible background interference, and tedious manual manipulation. Therefore, we propose DMF-Bimol that leverages addressable digital microfluidics to automate digital counting of single-cell mRNA and protein based on proximity ligation assay (PLA) and one-step RT-droplet digital PCR (RT-ddPCR). Through an engineered hydrophilic-hydrophobic interface, DMF-Bimol enables efficient single-cell isolation and lossless protein and nucleic acid processing. The closed droplet reaction system enhances the protein concentration and isolates exogenous contaminants, thereby dramatically improving the efficiency of the PLA reaction. The limit of detection of this approach achieves 3313 protein copies, marking a significant 17-fold enhancement in sensitivity over traditional benchtop PLA. This heightened sensitivity also uncovers a lower correlation between mRNA and protein levels in individual cells (Spearman r = 0.255) than bulk results, reflecting the complex relationship in heterogeneous cells. Using DMF-Bimol, we observed a significant upsurge of CD147 protein in CD138+ myeloma cells but consistent levels of CD147 mRNAs compared with normal leukocytes. This discovery indicates a possible consequence of CD147 oncogenic activation that tends to harness protein translation to bolster tumor cell survival and enhance invasiveness, highlighting the potential of DMF-Bimol in unveiling intricate dynamics in translation processes at the single-cell level.

Third Metal Ion Dictates the Catalytic Activity of the Two-Metal-Ion Pre-Ribosomal RNA-Processing Machinery.

Nucleic acid processing enzymes use a two-Mg2+-ion motif to promote the formation and cleavage of phosphodiester bonds. Yet, recent evidence demonstrates the presence of spatially conserved second-shell cations surrounding the catalytic architecture of proteinaceous and RNA-dependent enzymes. The RNase mitochondrial RNA processing (MRP) complex, which cleaves the ribosomal RNA (rRNA) precursor at the A3 cleavage site to yield mature 5'-end of 5.8S rRNA, hosts in the catalytic core one atypically-located Mg2+ ion, in addition to the ions forming the canonical catalytic motif. Here, we employ biased quantum classical molecular dynamics simulations of RNase MRP to discover that the third Mg2+ ion inhibits the catalytic process. Instead, its displacement in favour of a second-shell monovalent K+ ion propels phosphodiester bond cleavage by enabling the formation of a specific hydrogen bonding network that mediates the essential proton transfer step. This study points to a direct involvement of a transient K+ ion in the catalytic cleavage of the phosphodiester bond and implicates cation trafficking as a general mechanism in nucleic acid processing enzymes and ribozymes.

The altered TBI fecal microbiome is stable and functionally distinct.

Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

An Ion Concentration Polarization Microplatform for Efficient Enrichment and Analysis of ctDNA.

Strategies for rapid, effective nucleic acid processing hold tremendous significance to the clinical analysis of circulating tumor DNA (ctDNA), a family of important markers indicating tumorigenesis and metastasis. However, traditional techniques remain challenging to achieve efficient DNA enrichment, further bringing about complicated operation and limited detection sensitivity. Here, we developed an ion concentration polarization microplatform that enabled highly rapid, efficient enrichment and purification of ctDNA from a variety of clinical samples, including serum, urine, and feces. The platform demonstrated efficiently separating and enriching ctDNA within 30 s, with a 100-fold improvement over traditional methods. Integrating an on-chip isothermal amplification module, the platform further achieved 100-fold enhanced sensitivity in ctDNA detection, which significantly eliminated false-negative results in the serum or urine samples due to the low abundance of ctDNA. Such a simple-designed platform offers a user-friendly yet powerful diagnosis technique with a wide applicability, ranging from early tumor diagnosis to infection screening.

Inflammatory Manifestations Associated With Gut Dysbiosis in Alzheimer's Disease.

Recent studies strongly suggest that gut microbiome can influence brain functions and contribute to the development of Alzheimer's disease (AD). However, reported changes in the gut microbiomes in AD patients from different countries are not similar, and more research is needed to reveal the relationships between human microbiomes and AD in diverse ethnic populations. There is also an assumption that microbiome-associated peripheral inflammation might drive the development of sporadic AD. This cross-sectional study is aimed at analyzing the gut microbial profile and exploring potential associations with blood cytokines and some clinical parameters among individuals diagnosed with Alzheimer's in Kazakhstan. Consistent with previous studies, we have found that the microbial landscape in AD reveals specific alterations in the gut microbiome. Specifically, the AD patient group showed a decreased Firmicutes/Bacteroidetes ratio. The differential abundance analysis highlighted a dysbiosis in the gut microbiota of AD patients, marked by a reduced presence of Bifidobacterium, particularly B. breve. In our study, AD patients' altered gut microbiota composition notably features an increased presence of Pseudomonadota like Phyllobacterium and inflammatory bacteria such as Synergistetes and the Christensenellaceae family. The metabolic profiling of the AD microbiome reveals a predominant presence of pathways related to sugar, carrier molecules, tetrapyrrole, pyrimidine biosynthesis, and nucleic acid processing. This analysis also highlighted a marked reduction in SCFA, carbohydrate, polysaccharide, polyamine, and myo-inositol degradation pathways. The increases in the proinflammatory cytokines IL-1a, IL-8, IL-17A, IL-12p40, TNF-β, MCP-1, IL-2, and IL-12p70 and the anti-inflammatory cytokines IL-10 and IL-13 were observed in AD patients. Key variables driving the separation of AD and controls include inflammatory markers (IL-1a and IL-8), growth factors (EGF), lipids (LDL), BMI, and gut microbes, like genus Tyzzerella and Turicibacter and species Parabacteroides distasonis and Bacteroides eggerthii. We have also demonstrated that almost all cytokines strongly correlate with serum adiponectin levels and specific microbial taxa in AD patients. Thus, our findings identify potential microbial and inflammatory signatures in an ethnically distinct cohort of AD patients. These could serve as AD biomarkers and microbiota-based therapeutic targets for treating AD.

Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.

2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology.

Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome

Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.

Functional characterization of a naphthalene-O-methyltransferase from Nocardia sp. CS682

Methylation plays important roles in biosynthesis, metabolism, signal transduction, detoxification, protein sorting and repair, and nucleic acid processing. Generally the methyltransferases transfer methyl groups in various natural products using S-adenosyl methionine (SAM) as a cofactor. In this study, we examined and functionally characterized ThnM3 (enzyme), by testing various substrates with different chemical structures. Among the tested substrates, 1,8-dihydroxynaphthalene was the best substrate for methylation. Whole-cell biotransformation was performed using the enzyme in engineered Escherichia coli to produce 8-methoxynaphthalene-1-ol, and 1,8-dimethoxynaphthalene derivatives of 1,8-dihydroxynaphthalene. The products were confirmed using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopic analyses. Therefore, this study is the first to amplify, express the thnM3 (gene), and functionally characterize theThnM3, which exhibits the regioselective modifications of 1,8-dihydroxynaphthalene.

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencingand molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with>95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

The Photophysics of Nucleic Acids: Consequences for the Emergence of Life

AbstractAbsorption of ultraviolet (UV) radiation can trigger a variety of photophysical and photochemical reactions in nucleic acids. In the prebiotic era, on the surface of the early Earth, UV light could have played a major role in the selection of the building blocks of life via a balance between synthetic and destructive pathways. As nucleic acid monomers assembled into polymers, their survival and facility for non‐enzymatic replication hinged on their photostability and the ability for self‐repair of lesions, e. g., by UV‐induced charge transfer. Such photoprocesses are known to be sequence‐dependent and could have led to an additional prebiotic selection of the genetic sequence pools available to the earliest life forms. This review summarizes the photophysical processes in nucleic acids upon the absorption of a UV photon and their implications for chemical and genetic selection at the emergence of life and the origin of translation.

Gene Expression in the Developing Seed of Wild and Domesticated Rice.

The composition and nutritional properties of rice are the product of the expression of genes in the developing seed. RNA-Seq was used to investigate the level of gene expression at different stages of seed development in domesticated rice (Oryza sativa ssp. japonica var. Nipponbare) and two Australian wild taxa from the primary gene pool of rice (Oryza meridionalis and Oryza rufipogon type taxa). Transcriptome profiling of all coding sequences in the genome revealed that genes were significantly differentially expressed at different stages of seed development in both wild and domesticated rice. Differentially expressed genes were associated with metabolism, transcriptional regulation, nucleic acid processing, and signal transduction with the highest number of being linked to protein synthesis and starch/sucrose metabolism. The level of gene expression associated with domestication traits, starch and sucrose metabolism, and seed storage proteins were highest at the early stage (5 days post anthesis (DPA)) to the middle stage (15 DPA) and declined late in seed development in both wild and domesticated rice. However, in contrast, black hull colour (Bh4) gene was significantly expressed throughout seed development. A substantial number of novel transcripts (38) corresponding to domestication genes, starch and sucrose metabolism, and seed storage proteins were identified. The patterns of gene expression revealed in this study define the timing of metabolic processes associated with seed development and may be used to explain differences in rice grain quality and nutritional value.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Identification of protein biomarkers of attenuation and immunogenicity of centrin or p27 gene deleted live vaccine candidates of Leishmania against visceral leishmaniasis

Currently, no licensed vaccine is available for human visceral leishmaniasis (VL), a fatal disease caused by the protozoan parasite Leishmania donovani. Two of our live attenuated L. donovani vaccine candidates, either deleted for Centrin1 (LdCen1−/−) or p27 gene (Ldp27−/−), that display reduced growth in macrophages were studied to be safe, immunogenic and protective against VL in various animal models. This report involves the identification of differentially expressed proteins, their related pathways and its underlying mechanism in the intracellular stage of these parasites, using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods. Out of 50–60 proteins, found to be differentially expressed in these mutant parasites, 36 were found to be common in both the parasites. Such proteins mainly belong to the functional categories viz. metabolic enzymes, chaperones and stress proteins, proteins involved in translation, processing and transport and proteins involved in nucleic acid processing. Proteins known to be host protective, like Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c, calreticulin and those responsible for inducing immune response, namely tubulins, DEAD box RNA helicases, HSP70 and tryparedoxin, have been detected to be modulated in these parasites. Such proteins could be predicted as biomarkers, with further scope of study for their role in growth attenuation. SignificanceThis study aims at predicting proteomic biomarkers of Leishmania parasite growth attenuation, that have immunomodulatory role in the disease leishmaniasis. Advanced studies could be helpful in establishing the role of these identified proteins in parasitic virulence and to predict the host interaction at molecular level. Also, these proteins could be exploited as attenuation markers during the development of genetically modified live attenuated parasites as vaccine candidates. These could be cross validated in varied species of Leishmania and other tyrpanosomatids for similar response towards identifying them as universal biomarkers of attenuation.

Life sciences discovery and technology highlights

Life sciences discovery and technology highlights

Identification of prognostic immune cells and potential immune-related markers in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the most common and deadly tumors worldwide. Immunotherapy has emerged as a promising strategy for HCC treatment, and understanding the immune microenvironment of HCC provides a theoretical basis for identifying new immune targets. However, the roles of immune components and their regulatory mechanisms in HCC require further clarified.MethodsBy analyzing HCC expression profiles from The Cancer Genome Atlas (TCGA) database, we depicted the proportion profile of immune cells for each sample using the software CIBERSORTx. Using R packages, we also characterized the distribution of differentially expressed genes (DEGs) in immune cells, calculated the correlation coefficient between immune cells and common DEGs, and analyzed their biology function by Gene-Ontology analysis.ResultsWe found that seven immune cell types were related to the overall survival of HCC patients, and identified 3,692 differentially expressed immune-related genes, predominantly functioning in nucleic acid processing and metabolism. Moreover, 14 DEGs were identified as common candidates related to immune cells and overall survival.ConclusionsOur study not only presents an overview of the immune features of the microenvironment of HCC, but also provides potential targets related to immune components.

A novel method for inward fluid displacement in centrifugal microdevices for highly integrated nucleic acid processing with long-term reagent storage

Rotationally-driven lab-on-a-disc (LoaD) microfluidic systems are among the most promising methods for realizing complex nucleic acid (NA) testing at the point-of-need (PoN). However, despite significant advancements in NA amplification methods, very few sample-to-answer centrifugal microfluidic platforms have been realized due, in part, to a lack of on-disc sample preparation. In many instances, NA extraction (NAE) and/or lysis must be performed off-disc using conventional laboratory equipment and methods, thus tethering the assay to centralized facilities. Omission of in-line cellular lysis and NAE can be partially attributed to the nature of centrifugally-driven fluidics. Since flow is directed radially outward relative to the center of rotation (CoR), the number of possible sequential unit operations is limited by the disc radius. To address this, we report a simple, practical, automatable, and easy-to-implement method for inward fluid displacement (IFD) compatible with downstream nucleic acid amplification tests (NAATs). This approach leverages carbon dioxide (CO2) gas generated from on-board acid-base neutralization to drive liquid from the disc periphery towards the CoR. Large architectural features or highly corrosive chemicals required in other approaches were replaced with safe-to-handle IFD reagents that maintained their reactivity for at least six months of storage on-disc. Further, spatiotemporal control over neutralization initiation and containment of the resultant pneumatic pressure head was reliably achieved using a single diode for both laser-actuated valve opening and channel sealing, which eliminated the need for manual intervention (e.g., taping over vents) required in other IFD methods. Following initial characterization via dye recovery studies, we demonstrated for the first time that CO2-driven displacement does not inhibit downstream NAATs; NAs isolated direct-from-swab on disc were compatible with both ‘gold standard’ polymerase chain reaction (PCR) techniques and loop-mediated isothermal amplification (LAMP). The IFD approach described here stands to significantly ease integration of an increased number of sequential on-board processes, including cellular lysis, nucleic acid extraction, amplification, and detection, to greatly lower barriers towards automatable sample-to-answer LoaDs amenable for use on-site operation by non-technical personnel.

Mitochondrial сomplexome of etiolated pea shoots

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

Use of an unnatural amino acid to map helicase/DNA interfaces via photoactivated crosslinking.

Formation of protein/nucleic acid complexes is essential for life. From DNA replication and repair to transcription and translation, myriad different proteins bind nucleic acids to execute their essential cellular functions. Our understanding of the mechanisms underlying recognition and processing of nucleic acids can be greatly informed by mapping protein domains and residues that form interfaces with their DNA or RNA targets. Here we describe a crosslinking protocol in which the unnatural amino acid p-benzoyl-l-phenylalanine (Bpa) integrated at selected sites within the PriA DNA helicase is used to map surfaces of the protein that interact with specific positions in a synthetic DNA replication fork in vitro.

Endonuclease V from the archaeon Thermococcus kodakarensis is an inosine-specific ribonuclease.

Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA that is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in the regulation of inosine-containing RNA.

Characterization of Host Cell Potential Proteins Interacting with OsHV-1 Membrane Proteins.

The interaction between viral membrane associate proteins and host cellular surface molecules should facilitate the attachment and entry of OsHV-1 into host cells. Thus, blocking the putative membrane proteins ORF25 and ORF72 of OsHV-1 with antibodies that have previously been reported to subdue OsHV-1 replication in host cells, especially ORF25. In this study, prey proteins in host hemocytes were screened by pull-down assay with recombinant baits ORF25 and ORF72, respectively. Gene Ontology (GO) analysis of these prey proteins revealed that most of them were mainly associated with binding, structural molecule activity and transport activity in the molecular function category. The protein–protein interaction (PPI) network of the prey proteins was constructed by STRING and clustered via K-means. For both ORF25 and ORF72, three clusters of these prey proteins were distinguished that were mainly associated with cytoskeleton assembly, energy metabolism and nucleic acid processing. ORF25 tended to function in synergy with actins, while ORF72 functioned mainly with tubulins. The above results suggest that these two putative membrane proteins, ORF25 and ORF72, might serve a role in the transport of viral particles with the aid of a cytoskeleton inside cells.