Triblock PolyA-Mediated Protein Biosensor Based on a Size-Matching Proximity Hybridization Analysis.

The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.

A multi-channel electrochemical biosensor based on polyadenine tetrahedra for the detection of multiple drug resistance genes.

Antimicrobial resistance poses a serious threat to human health due to the high morbidity and mortality caused by drug-resistant microbial infections. Therefore, the development of rapid, sensitive and selective identification methods is key to improving the survival rate of patients. In this paper, a sandwich-type electrochemical DNA biosensor based on a polyadenine-DNA tetrahedron probe was constructed. The key experimental conditions were optimized, including the length of polyadenine, the concentration of the polyadenine DNA tetrahedron, the concentration of the signal probe and the hybridization time. At the same time, poly-avidin-HRP80 was used to enhance the electrochemical detection signal. Finally, excellent biosensor performance was achieved, and the detection limit for the synthetic DNA target was as low as 1 fM. In addition, we verified the practicability of the system by analyzing E. coli with the MCR-1 plasmid and realized multi-channel detection of the drug resistance genes MCR-1, blaNDM, blaKPC and blaOXA. With the ideal electrochemical interface, the polyA-based biosensor exhibits excellent stability, which provides powerful technical support for the rapid detection of antibiotic-resistant strains in the field.

A proximity-tagging system to identify membrane protein-protein interactions.

The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

Multi-omics data integration using ratio-based quantitative profiling with Quartet reference materials.

Characterization and integration of the genome, epigenome, transcriptome, proteome and metabolome of different datasets is difficult owing to a lack of ground truth. Here we develop and characterize suites of publicly available multi-omics reference materials of matched DNA, RNA, protein and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters. These references provide built-in truth defined by relationships among the family members and the information flow from DNA to RNA to protein. We demonstrate how using a ratio-based profiling approach that scales the absolute feature values of a study sample relative to those of a concurrently measured common reference sample produces reproducible and comparable data suitable for integration across batches, labs, platforms and omics types. Our study identifies reference-free 'absolute' feature quantification as the root cause of irreproducibility in multi-omics measurement and data integration and establishes the advantages of ratio-based multi-omics profiling with common reference materials.

The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes.

Mitochondria and peroxisomes are two types of functionally close-related organelles, and both play essential roles in lipid and ROS metabolism. However, how they physically interact with each other is not well understood. In this study, we apply the proximity labeling method with peroxisomal proteins and report that mitochondrial protein mitofusins (MFNs) are in proximity to peroxisomes. Overexpression of MFNs induces not only the mitochondria clustering but also the co-clustering of peroxisomes. We also report the enrichment of MFNs at the mitochondria-peroxisome interface. Induced mitofusin expression gives rise to more mitochondria-peroxisome contacting sites. Furthermore, the tethering of peroxisomes to mitochondria can be inhibited by the expression of a truncated MFN2, which lacks the transmembrane region. Collectively, our study suggests MFNs as regulators for mitochondria-peroxisome contacts. Our findings are essential for future studies of inter-organelle metabolism regulation and signaling, and may help understand the pathogenesis of mitofusin dysfunction-related disease.

A sphingolipid-mTORC1 nutrient-sensing pathway regulates animal development by an intestinal peroxisome relocation-based gut-brain crosstalk.

The mTOR-dependent nutrient-sensing and response machinery is the central hub for animals to regulate their cellular and developmental programs. However, equivalently pivotal nutrient and metabolite signals upstream of mTOR and developmental-regulatory signals downstream of mTOR are not clear, especially at the organism level. We previously showed glucosylceramide (GlcCer) acts as a critical nutrient and metabolite signal for overall amino acid levels to promote development by activating the intestinal mTORC1 signaling pathway. Here, through a large-scale genetic screen, we find that the intestinal peroxisome is critical for antagonizing the GlcCer-mTORC1-mediated nutrient signal. Mechanistically, GlcCer deficiency, inactive mTORC1, or prolonged starvation relocates intestinal peroxisomes closer to the apical region in a kinesin- and microtubule-dependent manner. Those apical accumulated peroxisomes further release peroxisomal-ß-oxidation-derived glycolipid hormones that target chemosensory neurons and downstream nuclear hormone receptor DAF-12 to arrest the animal development. Our data illustrate a sophisticated gut-brain axis that predominantly orchestrates nutrient-sensing-dependent development in animals.

Identification of a Small Probe That Can Be Conjugated to Proteins by Proximity Labeling.

Proximity labeling has been used to study protein-protein interactions and can also be used as a protein labeling tool. We developed a novel 14-amino acid peptide substrate for the proximity-labeling enzyme PafA. The N terminus of the peptide can be modified with biotin or fluorophores, which allows various chemical moieties to be ligated to the target protein. We used PafA-mediated peptide labeling to label antibodies with a biotin tag without affecting the antigen binding capacity of the antibody. Similar strategies can be used for other types of protein labeling in the future.