Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening.

UNLABELLED: Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE: This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

Simple Synthesis of Functionalized Paramagnetic Beads for Nucleic Acid Purification and Manipulation.

The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology ( Oberacker et al., 2019 ). Updated protocols can be found on the associated webpage (https://bomb.bio).