分子生物学

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

In many plant species, self-incompatibility (SI) is a mechanism that inhibits inbreeding. SI is gametophytic in the Solanaceae, with specificity determined by S-ribonucleases (S-RNases) in the pistil and S-locus F-box proteins (SLFs) in the pollen. The role of these proteins has been studied extensively in the Solanaceae, often using Petunia as a model. Using degenerate PCR and Sanger sequencing, this protocol identified three SLF sequences from self-incompatible diploid potato (Solanum okadae). While SLFs are well-characterized in model species like Petunia, there is limited sequence data and no standardized protocols for identifying SLFs in non-model species such as S. okadae, hindering broader insights into SI across the Solanaceae. This protocol fills that gap by using degenerate PCR and Sanger sequencing with primers designed from conserved Petunia SLF regions to identify SLF sequences in S. okadae. SLF sequences from 10 distinct Solanaceae members sharing maximum identity with the S2-haplotype of Petunia were used to design two pairs of primers targeting different regions of the target sequence. PCR amplification using designed degenerate primers yielded amplicons that were directly sequenced and joined together to get the partial SLF sequence. It was observed that the S. okadae shared an orthologous relation with the Petunia SLF according to the phylogenetic analysis. These SLFs could be used in future SI breakdown experiments via the competitive interaction route. This protocol, including the primer design, is novel for detecting SLF sequences in S. okadae.

We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes.

Inteins are elements translated within host proteins and removed via a unique protein splicing reaction. In this process, the two peptide bonds flanking the intein are rearranged, releasing the intein and leaving a standard peptide bond in its place. Due to their ability to shuffle peptide bonds in a specific and controlled manner, inteins have proven valuable in protein engineering, leading to the development of numerous impactful technologies. In one application, intein-based biosensors link the activity of a host protein to intein excision. Recently, we developed a biosensor to measure protein stability in vivo, in which the removal of an intein-protein fusion is required for antibiotic resistance. In our protocol, cells expressing our biosensor are logarithmically diluted and spotted on agar plates containing increasing levels of antibiotics. Following incubation, quantitative survival curves can be generated. We also developed a dual protein stability sensor where both antibiotic resistance and fluorescence can be used as readouts and demonstrated that co-expression of the chaperonin GroEL can promote survival and fluorescence. Taken together, our novel intein-based biosensor adds to the available tools to measure protein stability within the cellular environment.

Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example—one of the most challenging viruses for generating viral cDNA clones because of the factors listed above.

In molecular diagnosis, DNA extraction kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Previous fast extraction protocols such as detergent-based ones allow fast DNA extraction for nucleic acid amplification tests (NAAT), mainly polymerase chain reaction (PCR). The use of NaOH (dense alkali) to rupture cells and nuclei and destabilize the conformation of DNases might alleviate shortages and costs while retaining enough robustness to treat complicated samples with minimal environmental and logistical footprint. Biological samples are hand-crushed using a pestle in 1.5 mL tubes with 360 μL of 0.2 M NaOH for 3–5 min and incubated at 75 °C for 10 min. For immediate use, 115.2 μL of 1 M Tris (pH 8) and 364.8 μL nuclease-free water are added, and the sample is vortexed for 10 s and spun at 10,000× g for 3 min; then, 700 μL is transferred to a clean microtube. Two serial dilutions follow, and all concentrations are used as templates for PCR. A refined, storable extract can be produced by adding 70 μL of HCl 1 M (instead of Tris-HCl) and one volume of cold isopropanol to the extract for standard precipitation. This method can increase throughput in emergencies by field deployment in resource-limited settings (RLS) or allow benchtop backup in cases of acquisition disruption or sample surge in established facilities. The crude extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing NAAT in resource-limited settings with low costs and waste footprint or during prolonged crises, where supply chain failures may occur. The refined version produces alcohol-precipitated nucleic acids, suitable for both immediate use and for storage or dispatch for spatiotemporally separate analysis while offering much better amplification quality with a small increase in time and minimal increase in expendables/chemicals needed.

Fluorescent protein biosensors (FPBs) that turn on—go from dark to bright upon binding their ligands—enable the detection of targets in living cells with high sensitivity and spatial localization. Several approaches exist for creating turn-on FPBs, most notably the method that gave rise to the GCaMP family of genetically encoded calcium indicators. However, it remains challenging to modify these sensors to recognize new ligands. We recently developed adaptable turn-on maturation (ATOM) biosensors, in which target recognition by a small binding domain triggers chromophore maturation in the fluorescent protein to which it is attached. ATOM sensors are advantageous because they are generalizable (by virtue of the monobody and nanobody binding domains) and modular (binding domains and fluorescent proteins of various colors can be mixed and matched for multiplexed imaging), capable of detecting endogenously expressed proteins, and able to function in subcellular compartments including the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria. The protocols herein detail how to design, clone, and screen new ATOM sensors for detecting targets of choice. The starting materials are the genes encoding for a monobody or nanobody and for a cyan, yellow, or red fluorescent protein. We also present general guidelines for creating ATOM sensors using binding domains other than nanobodies and monobodies.

The early detection of meningitis pathogens—including Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella pneumoniae—through point-of-care (POC) systems is essential for mitigating the risk of neurological damage, enhancing patient outcomes, and facilitating prompt clinical decision-making. Nucleic acid amplification testing (NAAT) is a promising tool for improving the diagnosis process of bacterial pathogens associated with brain inflammation. This is due to its high sensitivity, rapidity, and compatibility with portable diagnostic platforms, making it particularly suitable for POC applications. This protocol introduces an innovative diagnostic approach designed to function effectively without the need for advanced laboratory equipment. By leveraging dual-priming isothermal amplification (DAMP), the assay uses custom internal primers to enhance specificity and minimize false results. Brilliant Green is used in this assay for fluorescence detection due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay demonstrated excellent specificity, absence of false positives, sensitivity comparable to loop-mediated isothermal amplification (LAMP), and a high S/B ratio.

Traditional approaches for the detection and differentiation of Bacillus cereus group species often face challenges due to the complexity of genetic discrimination between species. In this protocol, we propose a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay incorporates a universal fluorescent reporter and four DNA binding fragments, three of which are responsible for “opening up” the folded rRNA while the fourth strand is responsible for detecting single nucleotide variation (SNV) with high selectivity. The binding of the DNM to 16S rRNA results in the formation of the 10-23 DNAzyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. The developed biplex assay enables the detection of B. thuringiensis 16S rRNA and B. mycoides at fluorescein and Cy5 channels, respectively. The protocol offers two detection options: one utilizing extracted total RNA and the other involving crude cell lysate. The latter enables a fast and straightforward detection after 1.5 h with a hands-on time of ~15 min. The new protocol may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis.

This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.