Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.

A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

Purification of DNA Nanoparticles Using Photocleavable Biotin Tethers.

The number of applications of self-assembled deoxyribonucleic acid (DNA) origami nanoparticles (DNA NPs) has increased drastically, following the development of a variety of single-stranded template DNA (ssDNA) that can serve as the scaffold strand. In addition to viral genomes, such as M13 bacteriophage and lambda DNAs, enzymatically produced ssDNA from various template sources is rapidly gaining traction and being applied as the scaffold for DNA NP preparation. However, separating fully formed DNA NPs that have custom scaffolds from crude assembly mixes is often a multistep process of first separating the ssDNA scaffold from its enzymatic amplification process and then isolating the assembled DNA NPs from excess precursor strands. Only then is the DNA NP sample ready for downstream characterization and application. In this work, we highlight a single-step purification of custom sequence- or M13-derived scaffold-based DNA NPs using photocleavable biotin tethers. The process only requires an inexpensive ultraviolet (UV) lamp, and DNA NPs with up to 90% yield and high purity are obtained. We show the versatility of the process in separating two multihelix bundle structures and a wireframe polyhedral architecture.

Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Biotin-tagged fluorescent probe for in situ visualization of γ-glutamyl transpeptidase in cancerous cells and tissues.

γ-Glutamyl transpeptidase (GGT), a cell-surface enzyme, is strongly implicated in mammalian malignancy growth and migration processes including human hepatocarcinogens. However, simply and conveniently detect of GGT on the cell membrane remains highly challenging. In this study, a biotin-tagged fluorescent probe Nap-biotin-glu was developed using glutamic acid, naphthalimide, and biotin as the reaction site, fluorescent reporter, and membrane-targeting group, which required only three steps. Colocalization fluorescence imaging and immunofluorescence analysis indicated that probe Nap-biotin-glu was successfully realized in situ visualizing of GGT on the cell membrane.Owing to the significant over-expressed GGT level in tumor, the probe was successfully applied to distinguish cancer tissues from adjacent normal tissues.

AviTrap: A novel solution to achieve complete biotinylation.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

An array of femtoliter wells for sensitive detection of copper using click chemistry.

Sensitive detection of copper ion (Cu2+), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu2+ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N3) via Cu+-catalyzed click chemistry. Cu+ in the click chemistry reaction was generated by reducing Cu2+ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-ß-galactosidase (SßG). The MBs complex was then suspended in ß-galactosidase substrate fluorescein-di-ß-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SßG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu2+ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu2+ was 1 nM without signal amplification, which was 103 times lower than that of traditional fluorescence detection assays.

ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification.

Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

Streptavidin-biotin system-mediated immobilization of a bivalent nanobody onto magnetosomes for separation and analysis of 3-phenoxybenzoic acid in urine.

The compound 3-phenoxybenzoic acid (3-PBA) is frequently utilized as a biomarker to detect exposure to various pyrethroids. In this study, a bivalent nanobody (Nb2) specifically targeting 3-PBA was biotinylated and immobilized onto streptavidin (SA)-modified bacterial magnetic nanoparticles (BMPs), resulting in the formation of BMP-SA-Biotin-Nb2 complexes. These complexes demonstrated remarkable stability when exposed to strongly acidic solutions (4 M HCl), methanol (80%), and high ionic strength (1.37 M NaCl). An immunoassay was subsequently developed utilizing BMP-SA-Biotin-Nb2 as the capture agent and 3-PBA-horseradish peroxidase as the detection probe. The immunoassay exhibited an IC50 value (half-maximum signal inhibition concentration) of 1.11 ng mL-1 for 3-PBA. To evaluate the accuracy of the assay, spiked sheep and cow urine samples (ranging from 3.0 to 240 ng mL-1) were analyzed. The quantitative recoveries ranged from 82.5% to 113.1%, which agreed well with the findings obtained using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, the BMP-SA-Biotin-Nb2-based immunoassay holds great promise for rapid monitoring of 3-PBA following acid dissociation.

Enrichment of DNA replication intermediates by EdU pull down.

Analysis of replication fork structures in electron microscopy (EM) can provide important mechanistic insights in DNA replication studies. A major challenge in this type of analysis is the paucity of replication intermediates. At any given time only a small fraction of the restriction fragments of genomic DNA will contain a replication fork. To address this issue, we have developed an EdU-pull-down procedure to enrich for replicating DNA. Cells are exposed to a brief pulse of EdU, a cleavable biotin moiety is attached to EdU by copper-catalyzed azide-alkyne cycloaddition (CuAAC), in conditions that minimize the damage to DNA. Biotinylated DNA is purified with streptavidin beads, in conditions that facilitate association of long DNA filaments. Finally, the DNA is eluted by cleaving the biotin moiety. This approach can enrich over 150-times for replicating DNA and about 50-times in replication fork structures, as verified by EM. This procedure could benefit analysis of replication intermediates in EM as well as other techniques for the study of replicating DNA.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

A multiple signal amplification photoelectrochemical biosensor based on biotin-avidin system for kanamycin sensing in fish and milk via synergism of g-C3N4 and Ru@SiO2.

BACKGROUND: The residues of kanamycin can accumulate in the human body for a long time and pose serious health risks, including hearing loss, kidney poisoning, and drug allergic reactions. Therefore, it is crucial to develop a rapid, highly sensitive, and low-cost method for detecting kanamycin residues in foods. However, the current methods have limitations such as low sensitivity, expensive instruments, and multiple steps, which make them impractical for use in resource-limited environments and emergencies. In this study, the creation of a multiple-signal amplification photoelectrochemical biosensor to address these aforementioned issues is discussed. RESULTS: Herein, we proposed a multiple signal amplification photoelectrochemical (PEC) biosensor based on carboxylated g-C3N4 and avidin functionalized Ru@SiO2 for the ultrasensitive detection of kanamycin. The carboxylated g-C3N4 was a highly efficient photoactive substance for amplifying photoelectric signals and a substrate for aptamer immobilization. The DOS and PDOS of g-C3N4 were studied by simulation, and the sensing mechanism of the probe at the molecular level was revealed. Meanwhile, using Ru@SiO2 as a signal amplifying unit, through the cooperative work between Ru@SiO2 and g-C3N4, the photoelectric signal could be double amplified to produce an excellent photocurrent response. Under optimized conditions, the photocurrent response of the PEC biosensor to kanamycin was obtained at concentrations from 0.1 nM to 1000 nM with a lower detection limit of 4.1052 × 10-11 mol L-1. This protocol demonstrates high sensitivity, brilliant specific recognition ability, excellent reproducibility, and acceptable stability. SIGNIFICANCE: The first combination of g-C3N4 and avidin-Ru@SiO2 as photocurrent materials greatly enhanced the sensitivity of the PEC biosensors. Moreover, the specificity and sensitivity of the PEC biosensor were further improved through the specific interaction between kanamycin and aptamer. The photoelectric conversion mechanism based on g-C3N4 and two pathways for enhancing the photocurrent by Ru(byp)32+ were proposed. Through simulations of the DOS and PDOS of g-C3N4, the sensing mechanism of the probe at the molecular level was revealed. Under the optimum conditions, the PEC biosensor exhibited a wide linear concentration range and a low detection limit.

Chemical reagents for the enrichment of modified peptides in MS-based identification.

Chemical reagents with special groups as enrichable handles have empowered the ability to label and enrich modified peptides. Here is an overview of different chemical reagents with affinity tags to isolate labeled peptides and the latest developments of enrichment strategies. Biotin is the most used affinity tag due to its high interaction with avidin. To decrease the unfavorable influence of biotin for its poor efficiency in ionization and fragmentation in downstream MS analysis, cleavable moieties were installed between the reactive groups and biotin to release labeled peptides from the biotin. To minimize the steric hindrance of biotin, a two-step method was developed, for which alkyne- or azide-tagged linkers were firstly used to label peptides and then biotin was installed through click chemistry. Recently, new linkers using a small phosphonic acid as the affinity tag for IMAC or TiO2 enrichment have been developed and successfully used to isolate chemically labeled peptides in XL-MS. A stable P-C instead of P-O bond was introduced to linkers to differentiate labeled and endogenous phosphopeptides. Furthermore, a membrane-permeable phosphonate-containing reagent was reported, which facilitated the study of living systems. Taking a cue from classic chemical reactions, stable metal-complex intermediates, including cobalt and palladium complexes, have been developed as peptide purification systems. Advanced enrichment strategies have also been proposed, such as the two-stage IMAC enrichment method and biotin-based two-step reaction strategy, allowing the reduction of unwanted peptides and improvements for the analysis of specific labeled peptides. Finally, future trends in the area are briefly discussed.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.