Functional analysis of nuclear receptor genes in molting and metamorphosis of the cigarette beetle, Lasioderma serricorne.

Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva-pupa-adult transitions and caused 64.44-100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67-95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 µL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

Whole-Cell Display of Phospholipase D in Escherichia coli for High-Efficiency Extracellular Phosphatidylserine Production.

Phospholipids are widely utilized in various industries, including food, medicine, and cosmetics, due to their unique chemical properties and healthcare benefits. Phospholipase D (PLD) plays a crucial role in the biotransformation of phospholipids. Here, we have constructed a super-folder green fluorescent protein (sfGFP)-based phospholipase D (PLD) expression and surface-display system in Escherichia coli, enabling the surface display of sfGFP-PLDr34 on the bacteria. The displayed sfGFP-PLDr34 showed maximum enzymatic activity at pH 5.0 and 45 °C. The optimum Ca2+ concentrations for the transphosphatidylation activity and hydrolysis activity are 100 mM and 10 mM, respectively. The use of displayed sfGFP-PLDr34 for the conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that nearly all the PC was converted into PS at the optimum conditions. The displayed enzyme can be reused for up to three rounds while still producing detectable levels of PS. Thus, Escherichia coli/sfGFP-PLD shows potential for the feasible industrial-scale production of PS. Moreover, this system is particularly valuable for quickly screening higher-activity PLDs. The fluorescence of sfGFP can indicate the expression level of the fused PLD and changes that occur during reuse.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Dual function of magnetic nanocomposites-based SERS lateral flow strip for simultaneous detection of aflatoxin B1 and zearalenone.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two mycotoxins that often co-occur in corn. A surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) that can simultaneously detect AFB1 and ZEN in corn samples was developed employing the core-interlayer-satellite magnetic nanocomposites (Fe3O4@PEI/AuMBA@AgMBA) as dual-functional SERS tags. Under the optimal conditions, the detection ranges of AFB1 and ZEN in corn samples were 0.1-10 µg/kg and 4-400 µg/kg, respectively. Moreover, the test results for two mycotoxins in contaminated corn samples employing the suggested SERS-LFIA was in line with those of the HPLC technique. In view of its satisfactory sensitivity, accuracy, precision and short testing time (20 min), the developed system has a promising application prospect in the on-site simultaneous detection of AFB1 and ZEN.

Effects of glutaraldehyde and povidone-iodine on apoptosis of grass carp liver and hepatocytes.

Since disinfectants are used all over the world to treat illnesses in people and other animals, they pose a major risk to human health. The comprehensive effects of disinfectant treatments on fish liver, especially the impacts on oxidative stress, toxicological effects, transcriptome profiles, and apoptosis, have not yet been fully analyzed. In the current investigation, healthy grass carp were exposed to 80 µg/L glutaraldehyde or 50 µg/L povidone-iodine for 30 days. First, the findings of enzyme activity tests demonstrated that the administration of glutaraldehyde could considerably increase oxidative stress by lowering T-SOD, CAT, and GPx and raising MDA. Furthermore, KEGG research revealed that exposure to glutaraldehyde and povidone-iodine stimulated the PPAR signal pathway. To further elucidate the transcriptome results, the relative expressions of related DEGs in the PPAR signal pathway were verified. Glutaraldehyde induced apoptosis in liver tissue of grass carp; however, it activated cytotoxicity and apoptosis in grass carp hepatocytes when exposed to glutaraldehyde or povidone-iodine. According to the current study, disinfectants can cause the impairment of the immune system, oxidative stress, and attenuation of the PPAR signal pathway in the liver of grass carp, making them detrimental as dietary supplements for grass carp, particularly in the aquaculture sector.

Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

Anomalous origin of the left suprarenal, inferior phrenic arteries and left ovarian artery in a human cadaver.

This report addresses three variants identified within a female cadaver. Specifically, these were an anomalous origin of the right suprarenal artery, an abnormal bilateral ovarian vein branch, and a arterial tortuosity of the left ovarian artery. Indeed, the cadaver evinced abnormal origins in the case of the middle suprarenal artery (MSA), right inferior phrenic artery (IPA), and the renal capsule artery (emanating from the right renal artery). The MSA and IPA shared a common trunk with the inferior suprarenal artery. It was additionally observed that the right ovarian vein anastomoses the branches from the right kidney posterior inferior along with those to the renal fat capsule. Abnormal origin was evident in the case of the left ovarian artery, and arterial tortuosity was apparent in the lower region of the vessels. This report addresses both the clinical import of these variations and their likely causes. In the subdiaphragmatic region, surgical success and prognosis may be impacted by such anomalies; accordingly surgeons must be aware of anatomical variants of the ovarian and suprarenal arteries.

Removal of methylene blue by acrylic polymer adsorbents loaded with magnetic iron manganese oxides: Synthesis, characterization, and adsorption mechanisms.

Dyes pose significant risks for aquatic environments and biological health in general owing to their non-biodegradable nature, carcinogenicity, and toxicity. The effective treatment of dye wastewater has become an important research topic. In this study, acrylic polymers (AP) loaded with magnetic iron manganese oxides (MIMO) (AP/MIMO) were prepared and used for the first time for the adsorption of methylene blue (MB). Carbon in AP/MIMO exists predominantly in the C-H and C-C forms, with its content reaching 50.7%. Oxygen and nitrogen in AP/MIMO exist mainly in the -CO- and -N-C forms, with contents of up to 41.5% and 73.3%, respectively. MB removal by AP/MIMO was consistent with the pseudo-second-order kinetic model (R2 = 0.99), equilibrium was achieved within 20 min, and the highest MB capacity of 2611.23 mg g-1 was predicted by the Langmuir isotherm model (R2 = 0.91-0.94). AP/MIMO exhibited excellent MB adsorption performance in the pH range of 4-10, with a removal efficiency higher than 99.0% (MB = 100 mL 1000 mg L-1; AP/MIMO = 50 mg). Thermodynamic indicators, such as positive entropy (ΔS0; 98.30 J⋅mol-1⋅K-1), negative Gibbs free energy (ΔG0; -29.40, -28.50, and -27.50 KJ⋅mol-1), and positive enthalpy (ΔH0; 2.30 KJ⋅mol-1), demonstrated that MB removal by AP/MIMO was autonomous, favorable, and endothermic. In addition, the integration of experimental results and theoretical calculations verified that electrostatic interactions were the primary mechanism for MB adsorption at carboxyl sites on AP/MIMO. The total interaction energy between AP and MB was -310.43 kJ⋅mol-1, and the electrostatic effect had a decisive contribution to the MB adsorption, with a value of up to -341.06 kJ⋅mol-1. AP and MB were most likely bound by -COO and S atoms. Overall, AP/MIMO exhibits high adsorption capacity and shows potential as a high-performance magnetic polymer for MB removal.

Bibliometric Analysis of the Knowledge Landscape of Periodontal Disease in Pregnancy: A Noteworthy Multidisciplinary Issue.

Background: Pregnant women are highly susceptible to periodontal disease due to changes in hormonal and immune levels, which places a huge burden on the healthcare system and requires multidisciplinary interventions. This study aimed to assess the scientific profile and research trends related to periodontal disease in pregnancy through a bibliometric approach. Methods: Publications about periodontal disease in pregnancy from 2000 to 2022 were extracted from Science Citation Index Expanded. The knowledge networks of countries, institutions, authors, journals, references, and keywords in this field were constructed using the Citespace, VOSviewer, Bibliometrix, and BIBLIOMETRIC.COM platforms. Furthermore, correlations between the characteristics of countries and the number or impact of publications were analyzed. Results: 1162 original studies and reviews were included. There was a trend toward increased publications and citations in this field. The United States had the highest academic productivity and impact by a significant margin, while correlation analyses indicated that economic power may correlate with national scientific activity. The University of North Carolina and Offenbacher S were the most influential institution and author, respectively, taking center stage in the collaborative networks. However, only several loose connections between countries or institutions were identified in the global collaborative network analysis. Six of the top ten most productive journals were in Q1 in the Journal Citation Report, and there was intensive interaction between different research subfields, such as immunology, molecular biology, and microbiology. Frontier topics were primarily clustered in two areas: (1) oral microbiology, such as microbiome, oral bacteria, and Fusobacterium nucleatum; and (2) public health, such as quality of life, pregnancy outcomes, oral health, obesity, and classification. Conclusion: Since 2000, periodontal disease in pregnancy is receiving increasingly widespread attention and is rapidly evolving at a multidisciplinary level. Oral microbiological pathogenesis and public health impact-related research deserve more exploration and may be the future direction of research. Enhanced Collaboration and interdisciplinary communication may further facilitate progress in this discipline.

ABHD12 contributes to tumorigenesis and sorafenib resistance by preventing ferroptosis in hepatocellular carcinoma.

Sorafenib induces ferroptosis, making it a useful treatment against advanced liver hepatocellular carcinoma (LIHC). However, sorafenib resistance is extremely common among LIHC patients. Here, we used a comprehensive approach to investigate the effects of ABHD12, which regulates tumorigenesis and sorafenib resistance in LIHC. We validated ABHD12 expression was upregulated in LIHC tissue, which correlated with worse overall survival and related to tumor size or stage. ABHD12 facilitated a pro-tumorigenic phenotype involving increased cell proliferation, migration, and clonogenicity as well as sorafenib resistance. Knockout of ABHD12 sensitized liver cancer cells to sorafenib-induced ferroptosis. Co-delivery of sorafenib and ABHD12 inhibitor into a nude mouse model enhanced therapeutic efficacy for LIHC. Our study demonstrates that ABHD12 contributes to tumor growth and sorafenib resistance in liver cancer, which indicate the promising potential of ABHD12 in diagnosis and prognosis as well as highlight the potential therapeutic applications for co-delivery of sorafenib and ABHD12 inhibitor.

TiO2 Nanocontainers Coconstructed Using Polymers and Corrosion Inhibitors for Anticorrosion Reinforcement of Waterborne Epoxy Coatings.

Stimulus-responsive coatings can provide active corrosion protection in response to environmental changes, but they have not reached their anticipated application prospects because of the intricate preparation processes of hollow materials and methods for loading corrosion inhibitors. Herein, polyaniline molybdate corrosion inhibitor and polydopamine-wrapped titanium dioxide nanocontainers (named TiO2/PANI-MoO42-/PDA) are synthesized via a simple three-step electrostatic assembly technique. Introducing TiO2/PANI-MoO42-/PDA nanocontainers in smart waterborne epoxy (WEP) coatings affords the latter with high barriers and long-term corrosion protection. The successful deposition of each layer on the TiO2 nanocontainer surface was validated via Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy. Release test results show that the molybdate corrosion inhibitor exhibits notable pH-responsive activity under acidic conditions and slow release in neutral environments, which improves the corrosion resistance of coatings. The addition of synthetic nanocontainers greatly improves the impermeability of WEP coatings. The charge transfer resistance of WEP/TiO2/PANI-MoO42-/PDA coatings is 1.79 × 1011 Ω cm2 after 30 day immersion in a 3.5 wt % NaCl solution, which is 3.32 × 105 times higher than that of WEP coatings. WEP/TiO2/PANI-MoO42-/PDA coatings remain uniform and reliable, even after 50 days of salt spray exposure. The excellent corrosion protection of WEP/TiO2/PANI-MoO42-/PDA coatings is attributed to (1) the enhanced dispersion and compatibility of PDA in the coating for nanocontainers, (2) the combination of phenolic hydroxyl groups of PDA and Fe, which inhibit corrosion activity on the exposed metal surface, and (3) the on-demand release of the MoO42- inhibitor, which provides sustained passivation protection. This work proposes a strategy to simplify the preparation of responsive long-term anticorrosion coatings and extend their service lives.

Improving the Activity of Tryptophan Synthetase via a Nucleic Acid Scaffold.

Tryptophan synthetase (TSase), which functions as a tetramer, is a typical enzyme with a substrate channel effect, and shows excellent performance in the production of non-standard amino acids, histamine, and other biological derivatives. Based on previous work, we fused a mutant CE protein (colistin of E. coli, a polypeptide with antibacterial activity) sequence with the sequence of TSase to explore whether its catalytic activity could be enhanced, and we also analyzed whether the addition of a DNA scaffold was a feasible strategy. Here, dCE (CE protein without DNase activity) protein tags were constructed and fused to the TrapA and TrapB subunits of TSase, and the whole cell was used for the catalytic reaction. The results showed that after the dCE protein tag was fused to the TrapB subunit, its whole cell catalytic activity increased by 50%. Next, the two subunits were expressed separately, and the proteins were bound in vitro to ensure equimolar combination between the two subunits. After the dCE label was fused to TrapB, the activity of TSase assembled with TrapA also improved. A series of experiments revealed that the enzyme fused with dCE9 showed higher activity than the wild-type protein. In general, the activity of assembly TSase was optimal when the temperature was 50 °C and the pH was about 9.0. After a long temperature treatment, the enzyme maintained good activity. With the addition of exogenous nucleic acid, the activity of the enzyme increased. The maximum yield was 0.58 g/L, which was almost three times that of the wild-type TSase (0.21 g/L). The recombinant TSase constructed in this study with dCE fusion had the advantages of higher heat resistance and higher activity, and confirmed the feasibility of adding a nucleic acid scaffold, providing a new idea for the improvement of structurally similar enzymes.

Targeting lung cancer with clinically relevant EGFR mutations using anti-EGFR RNA aptamer.

A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current landscape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most common oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispecific aptamer (bsApt) showed superior labeling of NSCLC cells in vitro relative to monospecific aptamers. However, dual targeting in vivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, biodistribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.

Bacterial Expression of Promelittin for Safe and Effective Tumor Therapy.

Bacteria-based tumor therapy has attracted much attention due to its unique mechanism and abundant application. With the rapid development of synthetic biology, utilizing gene technology to make bacteria express therapeutic agents has greatly innovated bacterial therapy paradigms. Herein, we constructed an Escherichia coli expressing promelittin protein system based on the Trojan horse strategy, which limited the toxicity of melittin through the fusion protein during melittin expression. After targeted colonization of bacteria in tumor tissues, promelittin was activated by matrix metalloproteinase, followed by causing tumor cell death through a membrane-lytic mechanism. Additionally, the released cytolytic melittin in turn killed the maternal bacteria, eliminating safety hazards and triggering host immunity. Detailed experiments revealed that the bacteria expressing the promelittin system could significantly inhibit the proliferation and metastasis of primitive tumors in a CT26-bearing mice model. This study sheds insights into the development of bacteria-based synergistic tumor therapy.

Mutation-derived, genomic instability-associated lncRNAs are prognostic markers in gliomas.

Background: Gliomas are the most commonly-detected malignant tumors of the brain. They contain abundant long non-coding RNAs (lncRNAs), which are valuable cancer biomarkers. LncRNAs may be involved in genomic instability; however, their specific role and mechanism in gliomas remains unclear. LncRNAs that are related to genomic instability have not been reported in gliomas. Methods: The transcriptome data from The Cancer Genome Atlas (TCGA) database were analyzed. The co-expression network of genomic instability-related lncRNAs and mRNA was established, and the model of genomic instability-related lncRNA was identified by univariate Cox regression and LASSO analyses. Based on the median risk score obtained in the training set, we divided the samples into high-risk and low-risk groups and proved the survival prediction ability of genomic instability-related lncRNA signatures. The results were verified in the external data set. Finally, a real-time quantitative polymerase chain reaction assay was performed to validate the signature. Results: The signatures of 17 lncRNAs (LINC01579, AL022344.1, AC025171.5, LINC01116, MIR155HG, AC131097.3, LINC00906, CYTOR, AC015540.1, SLC25A21.AS1, H19, AL133415.1, SNHG18, FOXD3.AS1, LINC02593, AL354919.2 and CRNDE) related to genomic instability were identified. In the internal data set and Gene Expression Omnibus (GEO) external data set, the low-risk group showed better survival than the high-risk group (P < 0.001). In addition, this feature was identified as an independent risk factor, showing its independent prognostic value with different clinical stratifications. The majority of patients in the low-risk group had isocitrate dehydrogenase 1 (IDH1) mutations. The expression levels of these lncRNAs were significantly higher in glioblastoma cell lines than in normal cells. Conclusions: Our study shows that the signature of 17 lncRNAs related to genomic instability has prognostic value for gliomas and could provide a potential therapeutic method for glioblastoma.

TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.

Applications of Programmable Endonucleases in Sequence- and Ligation-Independent Seamless DNA Assembly.

Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of the target DNA with the guide RNA or DNA to cleave DNA strands. Therefore, they are capable of recognizing and cleaving DNA sequences at virtually any arbitrary site. The present review focuses on the commonly used in vivo and in vitro recombination-based gene cloning methods and the application of programmable endonucleases in these sequence- and ligation-independent DNA assembly methods. The advantages and shortcomings of the programmable endonucleases utilized as tools for gene cloning are also discussed in this review.

A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia.

Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.

The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.