A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.

Sequence-Specific Detection of DNA Strands Using a Solid-State Nanopore Assisted by Microbeads

Simple, rapid, and low-cost detection of DNA with specific sequence is crucial for molecular diagnosis and therapy applications. In this research, the target DNA molecules are bonded to the streptavidin-coated microbeads, after hybridizing with biotinylated probes. A nanopore with a diameter significantly smaller than the microbeads is used to detect DNA molecules through the ionic pulse signals. Because the DNA molecules attached on the microbead should dissociate from the beads before completely passing through the pore, the signal duration time for the target DNA is two orders of magnitude longer than free DNA. Moreover, the high local concentration of target DNA molecules on the surface of microbeads leads to multiple DNA molecules translocating through the pore simultaneously, which generates pulse signals with amplitude much larger than single free DNA translocation events. Therefore, the DNA molecules with specific sequence can be easily identified by a nanopore sensor assisted by microbeads according to the ionic pulse signals.

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

SummaryBiotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Pseudoscorpion Wolbachia symbionts: Diversity and Evidence for a New Supergroup S

Background Wolbachia are the most widely spread endosymbiotic bacteria, present in a wide variety of insects and two families of nematodes, but as of now, relatively little genomic data has been available. The Wolbachia symbiont can be parasitic, as described for many arthropod systems, an obligate mutualist, as in filarial nematodes or a combination of both in some organisms. They are currently classified into 16 monophyletic lineage groups ("supergroups"). Although the nature of these symbioses remains largely unknown, expanded Wolbachia genomic data will contribute to understanding their diverse symbiotic mechanisms and evolution. Results This report focuses on Wolbachia infections in three pseudoscorpion species infected by two distinct groups of Wolbachia strains , based upon multi-locus phylogenies. Geogarypus minor harbours w Gmin and Chthonius ischnocheles harbours w Cisc, both closely related to supergroup H, while Atemnus politus harbours w Apol, a member of a novel supergroup S along with Wolbachia from the pseudoscorpion Cordylochernes scorpioides ( w Csco), most closely related to Wolbachia supergroups C and F. Using target enrichment by hybridization with Wolbachia -specific biotinylated probes to capture large fragments of Wolbachia DNA, we produced two draft genomes of w Apol. Annotation of w Apol highlights presence of a biotin operon, which is incomplete in many sequenced Wolbachia genomes. Conclusions The present study highlights at least two symbiont acquisition events among pseudoscorpion species. Phylogenomic analysis indicates that the Wolbachia from Atemnus politus ( w Apol), forms a separate supergroup ("S") with the Wolbachia from Cordylochernes scorpioides (w Csco). Interestingly, the biotin operon, present in w Apol, appears to have been horizontally transferred multiple times along Wolbachia evolutionary history.

Pseudoscorpion Wolbachia symbionts: Diversity and Evidence for a New Supergroup S

Background Wolbachia are the most widely spread endosymbiotic bacteria, present in a wide variety of insects and two families of nematodes, but as of now, relatively little genomic data has been available. The Wolbachia symbiont can be parasitic, as described for many arthropod systems, an obligate mutualist, as in filarial nematodes or a combination of both in some organisms. They are currently classified into 16 monophyletic lineage groups ("supergroups"). Although the nature of these symbioses remains largely unknown, expanded Wolbachia genomic data will contribute to understanding their diverse symbiotic mechanisms and evolution. Results This report focuses on Wolbachia infections in three pseudoscorpion species infected by two distinct groups of Wolbachia strains , based upon multi-locus phylogenies. Geogarypus minor harbours w Gmin and Chthonius ischnocheles harbours w Cisc, both closely related to supergroup H, while Atemnus politus harbours w Apol, a member of a novel supergroup S along with Wolbachia from the pseudoscorpion Cordylochernes scorpioides ( w Csco), most closely related to Wolbachia supergroups C and F. Using target enrichment by hybridization with Wolbachia -specific biotinylated probes to capture large fragments of Wolbachia DNA, we produced two draft genomes of w Apol. Annotation of w Apol highlights presence of a biotin operon, which is incomplete in many sequenced Wolbachia genomes. Conclusions The present study highlights at least two symbiont acquisition events among pseudoscorpion species. Phylogenomic analysis indicates that the Wolbachia from Atemnus politus ( w Apol), forms a separate supergroup ("S") with the Wolbachia from Cordylochernes scorpioides (w Csco). Interestingly, the biotin operon, present in w Apol, appears to have been horizontally transferred multiple times along Wolbachia evolutionary history.

Enhanced Immune Response Against the Thomsen-Friedenreich Tumor Antigen Using a Bivalent Entirely Carbohydrate Conjugate

The Thomsen-Friedenreich (TF) antigen is a key target for the development of anticancer vaccines, and this ongoing challenge remains relevant due to the poor immunogenicity of the TF antigen. To overcome this challenge, we adopted a bivalent conjugate design which introduced both the TF antigen and the Thomsen-nouveau (Tn) antigen onto the immunologically relevant polysaccharide A1 (PS A1). The immunological results in C57BL/6 mice revealed that the bivalent, Tn-TF-PS A1 conjugate increased the immune response towards the TF antigen as compared to the monovalent TF-PS A1. This phenomenon was first observed with enzyme-linked immunosorbent assay (ELISA) where the bivalent conjugate generated high titers of IgG antibodies where the monovalent conjugate generated an exclusive IgM response. Fluorescence-activated cell sorting (FACS) analysis also revealed increased binding events to the tumor cell lines MCF-7 and OVCAR-5, which are consistent with the enhanced tumor cell lysis observed in a complement dependent cytotoxicity (CDC) assay. The cytokine profile generated by the bivalent construct revealed increased pro-inflammatory cytokines IL-17 and IFN-γ. This increase in cytokine concentration was matched with an increase in cytokine producing cells as observed by ELISpot. We hypothesized the mechanisms for this phenomenon to involve the macrophage galactose N-acetylgalactosamine specific lectin 2 (MGL2). This hypothesis was supported by using biotinylated probes and recombinant MGL2 to measure carbohydrate-protein interactions.

Pseudoscorpion Wolbachia symbionts: Diversity and Evidence for a New Supergroup S

Background Wolbachia are the most widely spread endosymbiotic bacteria, present in a wide variety of insects and two families of nematodes, but so far, relatively little genomic data is available. The Wolbachia symbiont can be a parasite, as described for many arthropods, or an obligate mutualist, as in filarial nematodes. Although, the nature of these symbioses remains largely unknown, diverse Wolbachia genomic data will contribute to understanding their diverse symbiotic mechanisms. Results Our study focuses on Wolbachia infections in pseudoscorpion species collected in Montpellier (France) and indicates two distinct groups of Wolbachia : Geogarypus minor harbors Wolbachia ( w Gmin) and Chthonius ischnocheles harbors Wolbachia ( w Cisc), both related to supergroup H and Atemnus politus harbours Wolbachia ( w Apol), forming a novel divergent clade with Wolbachia from the pseudoscorpion Cordylochernes scorpioides . This later clade forms a new supergroup S, most closely related to Wolbachia supergroups C and F. Our data suggest multiple symbiont acquisition events within the evolutionary history of pseudoscorpions. Using target enrichment by hybridization with Wolbachia -specific biotinylated probes to capture large fragments of Wolbachia DNA, we produced a draft genome of w Apol characterized by a moderate Wolbachia size (1,445,964bp) containing a moderate number of transposable elements and WO bacteriophage insertions (total of 77,522 bp). Conclusions Our analyses indicate that w Apol forms a diverget clade. Annotation highlights complete biochemical pathways which are incomplete in many sequenced Wolbachia genomes, such as vitamin B and the cytochrome bd ubiquinol oxidase pathway. Further, the biotin operon appears to have been horizontally transferred multiple times along the Wolbachia evolutionary history.