Human RNase Research Articles

A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA Decay.

Eukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

B-192 Covid on the PCR Pro: a consumer-friendly PCR test

Abstract Background The emergence of COVID-19 highlighted the need for rapid and accurate diagnostic solutions that can be deployed on a broad scale. The complexity and long turn-around-time of PCR testing typically performed in molecular laboratories is not compatible with community-wide screening. To overcome these challenges, we present the Co-Dx PCR Pro™, a rapid and simple test platform developed for self-collection and at-home use. The instrument is controlled from the user’s smartphone or tablet and performs sample treatment, RT-PCR, and DNA melting analysis. An automatic calling algorithm provides a simple, “Detected,” or, “Not detected,” result. We demonstrate the utility of this platform using a test for the detection of SARS-COV-2. Methods A self-collected anterior nasal swab sample and 2 mLs of water are added to the collection cup. Closing the lid of the cup initiates rehydrating a dried Proteinase K mixture to digest proteins in the sample. After a brief Proteinase K heat-kill step, the instrument mixes the sample with a dried PCR master mix. Primers target two SARS-COV-2 specific genes (RdRp and E gene) and the human RNase P gene as a control. The sample undergoes an RT step followed by 45 cycles of PCR and DNA melting analysis. Detection is by an automatic calling algorithm that identifies a SARS-COV-2 amplicon melting peak at 82°C and RNase P peak at 87°C. The total runtime is less than 30 minutes. The limit of detection for the assay was assessed by contriving intact virus in negative sample matrix, which was then applied to dry swabs. In addition, frozen and banked clinical samples were tested using the Logix Smart®™ Covid-19 test as a comparator assay. A third party performed a usability assessment by having (1) naïve users follow the instructions and self-administer the test (2) expert trained users perform an independent assessment of the assay. Participants were then asked to complete a usability assessment. Results The 95% limit of detection was 2,100 copies/swab using contrived nasal swabs. Clinical samples were blinded to the operator and run on the platform. All samples resulted in a diagnosis concordant with the comparator assay. The usability assessment was performed by six naïve users and resulted in a systems usability scale score of “Best” (84.2 ± 14.3, out of 100). The two expert users determined an 80% limit of detection of 700 genome equivalents and “ease of use,” and “satisfaction,” scores of 4.5/5.0 and 5.0/5.0, respectively. Conclusions Performance of the PCR Pro platform is on par with laboratory-based PCR testing. The benefits of our assay are its speed and the low complexity platform accessible to the lay. This platform could improve access to molecular diagnostic testing, including use at home. Future expansion to include common respiratory pathogens (Flu, RSV, Strep) will increase utility. Additionally, anonymized results can be aggregated and provide a wealth of epidemiological information at the community level to better inform public health initiatives and health outcomes.

RNA-DNA hybrids on protein coding genes are stabilized by loss of RNase H and are associated with DNA damages during S-phase in fission yeast.

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.

Impact of relative humidity on SARS-CoV-2 RNA extraction using Nextractor automated extraction system.

This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity's impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced shortpalindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

Evaluation of RNase therapy in systemic lupus erythematosus: a randomised phase 2a clinical trial of RSLV-132

BackgroundCirculating, extracellular RNA is the primary trigger of type I interferon in systemic lupus erythematosus (SLE), and interferon is known to play a central pathogenic role in the disease. RSLV-132...

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli.

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

Engineering Human Pancreatic RNase 1 as an Immunotherapeutic Agent for Cancer Therapy Through Computational and Experimental Studies.

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.

Selective Characterization of mRNA 5' End-Capping by DNA Probe-Directed Enrichment with Site-Specific Endoribonucleases.

The N7-methyl guanosine cap structure is an essential 5' end modification of eukaryotic mRNA. It plays a critical role in many aspects of the life cycle of mRNA, including nuclear export, stability, and translation. Equipping synthetic transcripts with a 5' cap is paramount to the development of effective mRNA vaccines and therapeutics. Here, we report a simple and flexible workflow to selectively isolate and analyze structural features of the 5' end of an mRNA by means of DNA probe-directed enrichment with site-specific single-strand endoribonucleases. Specifically, we showed that the RNA cleavage by site-specific RNases can be effectively steered by a complementary DNA probe to recognition sites downstream of the probe-hybridized region, utilizing a flexible range of DNA probe designs. We applied this approach using human RNase 4 to isolate well-defined cleavage products from the 5' end of diverse uridylated and N1-methylpseudouridylated mRNA 5' end transcript sequences. hRNase 4 increases the precision of the RNA cleavage, reducing product heterogeneity while providing comparable estimates of capped products and their intermediaries relative to the widely used RNase H. Collectively, we demonstrated that this workflow ensures well-defined and predictable 5' end cleavage products suitable for analysis and relative quantitation of synthetic mRNA 5' cap structures by UHPLC-MS/MS.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics.

We have developed RHINO, a genetically encoded sensor that selectively binds RNA:DNA hybrids enabling live-cell imaging of cellular R-loops. RHINO comprises a tandem array of three copies of the RNA:DNA hybrid binding domain of human RNase H1 connected by optimized linker segments and fused to a fluorescent protein. This tool allows the measurement of R-loop abundance and dynamics in live cells with high specificity and sensitivity. Using RHINO, we provide a kinetic framework for R-loops at nucleoli, telomeres and protein-coding genes. Our findings demonstrate that R-loop dynamics vary significantly across these regions, potentially reflecting the distinct roles R-loops play in different chromosomal contexts. RHINO is a powerful tool for investigating the role of R-loops in cellular processes and their contribution to disease development and progression.

A-259 Detecting Human RNase P Gene Prevents False-Negative of SARS-CoV-2

Abstract Background Real-time Reverse transcriptase—polymerase chain reaction (RT-PCR) is a regular technique for detecting SARS-CoV-2 infection. Human RNase P sequence is using as internal control for PCR assay usually. This article discusses the correlation between SARS-CoV-2 PCR result and human RNase P gene result. Methods Nasopharyngeal swabs were collected from two patients within three consecutive days. We detected for SARS-CoV-2 target gene and human RNase P gene by using real-time RT-PCR assay, which SARS-CoV-2 target gene includes E gene, Rarp gene and N gene . And the threshold cycle values (Ct) were determined by cobas® z 480. Evaluate the results of SARS-CoV-2 and RNase P gene detection. All samples were also analysis for SARS-CoV-2 infection in Taiwan Centers for Disease Control. Results Results of SARS-CoV-2 PCR assay were consistent in both laboratories. SARS-CoV-2 target genes were detected from specimens of both patients on day1. Ct values of E gene were 29.63 and 29.50. Ct values of Rdrp gene were 32.23 and 32.50. Ct values of N gene were 33.09 and 32.68. Ct values of RNase P gene were 21.21 and 21.22. On day 2 and 3, all specimens were negative detected for SARS-CoV-2 PCR. Ct values of RNase P gene were 25.73 and 26.16 on day 2 and 29.82 and 30.28 on day 3. Conclusion The Ct values of human RNase P gene detection can be correlated with the numbers of collected cells. Comparing the results of two patients for three consecutive days, the Ct value of human RNase P gene related to SARS-CoV-2 PCR result. Collection technique can be a factor to affect Ct value of human RNase P gene detection. Develop acceptance range for Ct value of RNase P gene detection can be a quality standard for sample collection to prevent false-negative of SARS-CoV-2.

Digital PCR using a simple PDMS microfluidic chip and standard laboratory equipment.

Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.

РОЛЬ РИБОНУКЛЕАЗ В ИММУНОПАТОГЕНЕЗЕ ЭКСПЕРИМЕНТАЛЬНОГО ОСТРОГО РЕСПИРАТОРНОГО ДИСТРЕСС-СИНДРОМА

Background. An adequate assessment of the pathogenesis and course of acute respiratory distress syndrome (ARDS), as well as the search for new methods of treating this pathology, are urgent tasks of modern medicine. The aim of the study was to reveal the influence of ribonuclease enzymes (RNases) on the pathological process of ARDS. Material and methods. Lipopolysaccharide from Escherichia Coli and Pseudomonas Aeruginosa, thromboplastincalcium mixture solution, and recombinant human RNASE2 protein were used to simulate ARDS in laboratory Wistar rats. Results. The resulting models of ARDS were characterized by persistent significant hemostasis disorders and an increase in the level of cytokines; a fragment of human ribonuclease had an impact on the change in the levels of thrombin time, activated partial thromboplastin time and interleukin-6 in the studied models. Conclusions. The changes observed in experimental animals indicate the influence of ribonucleases on the course of the pathological process in ARDS, and the presented models allow us to evaluate the relationship of the selected factors with various outcomes.

Human RNase 1 can extensively oligomerize through 3D domain swapping thanks to the crucial contribution of its C-terminus

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory “pancreatic-type” (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers.Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases.Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.

Targeting Ribonucleases with Small Molecules and Bifunctional Molecules.

Ribonucleases (RNases) cleave and process RNAs, thereby regulating the biogenesis, metabolism, and degradation of coding and noncoding RNAs. Thus, small molecules targeting RNases have the potential to perturb RNA biology, and RNases have been studied as therapeutic targets of antibiotics, antivirals, and agents for autoimmune diseases and cancers. Additionally, the recent advances in chemically induced proximity approaches have led to the discovery of bifunctional molecules that target RNases to achieve RNA degradation or inhibit RNA processing. Here, we summarize the efforts that have been made to discover small-molecule inhibitors and activators targeting bacterial, viral, and human RNases. We also highlight the emerging examples of RNase-targeting bifunctional molecules and discuss the trends in developing such molecules for both biological and therapeutic applications.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Recombinase polymerase amplification (RPA) running at 37–42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.