Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells.

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid-modified 2,3, L-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-ß-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of ß-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of ß-galactosidase was detected in the membrane and vesicles of the cells.

Learning Protein-Ligand Unbinding Pathways via Single-Parameter Community Detection.

Understanding the dynamics of biomolecular complexes, e.g., of protein-ligand (un)binding, requires the comprehension of paths such systems take between metastable states. In MD simulations, paths are usually not observable per se, but they need to be inferred from simulation trajectories. Here, we present a novel approach to cluster trajectories based on a community detection algorithm that necessitates only the definition of a single parameter. The unbinding of the streptavidin-biotin complex is used as a benchmark system and the A2a adenosine receptor in complex with the inhibitor ZM241385 as an elaborate application. We demonstrate how such clusters of trajectories correspond to pathways and how the approach helps in the identification of reaction coordinates for a considered (un)binding process.

Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification for reinforced serum microRNA probing.

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.

In vivo-stable bis-iminobiotin for targeted radionuclide delivery with the mutant streptavidin.

Targeted delivery of radionuclides to tumors is significant in theranostics applications for precision medicine. Pre-targeting, in which a tumor-targeting vehicle and a radionuclide-loaded effector small molecule are administered separately, holds promise since it can reduce unnecessary internal radiation exposure of healthy cells and can minimize radiation decay. The success of the pre-targeting delivery requires an in vivo-stable tumor-targeting vehicle selectively binding to tumor antigens and an in vivo-stable small molecule effector selectively binding to the vehicle accumulated on the tumor. We previously reported a drug delivery system composed of a low-immunogenic streptavidin with weakened affinity to endogenous biotin and a bis-iminobiotin with high affinity to the engineered streptavidin. It was, however, unknown whether the bis-iminobiotin is stable in vivo when administered alone for the pre-targeting applications. Here we report a new in vivo-stable bis-iminobiotin derivative. The keys to success were the identification of the degradation site of the original bis-iminobiotin treated with mouse plasma and the structural modification of the degradation site. We disclosed the successful pre-targeting delivery of astatine-211 (211At), α-particle emitter, to the CEACAM5-positive tumor in xenograft mouse models.

Advancing MicroRNA Detection: Enhanced Biotin-Streptavidin Dual-Mode Phase Imaging Surface Plasmon Resonance Aptasensor.

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.

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.

Modified streptavidin-biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤ 35, and Ct ≤ 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

Drug-Loaded Bacillus Calmette-Guérin Bacteria for Immuno-Chemo Combo Therapy in Bladder Cancer.

Intravesical Bacillus Calmette-Guérin (BCG) is a well-established strategy for managing high-risk nonmuscle-invasive bladder cancer (NMIBC); however, over half of patients still experience disease recurrence or progression. Although the combined intravesical instillation of various chemotherapeutic drugs is implemented in clinical trials to enhance the BCG therapy, the outcome is far from satisfying due to severe irritative effects and treatment intolerance at high doses. Therefore, it is adopted the "biotin-streptavidin strategy" to doxorubicin (DOX)-encapsulated nanoparticles within live BCG bacteria (DOX@BCG) to improve treatment outcomes. Adherence of BCG to the bladder epithelium helps precisely target DOX@BCG to the local tumor cells and simultaneously increases intratumoral transport of therapeutic drugs. DOX@BCG effectively inhibits cancer progression and prolongs the survival of rats/mice with orthotopic bladder cancer owing to synergism between BCG-immunotherapy, DOX-chemotherapy, and DOX-induced immunogenic tumor cell death; furthermore, it exhibits improved tolerance and biosafety, and establishes antitumor immunity in the tumor microenvironment. Therefore, the drug-loaded live BCG bacterial delivery system holds considerable potential for clinical translation in the intravesical treatment of bladder cancer.

Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 µM) underlines potential interest of this nanoplatform for biomedical applications.

Development of gold nanocluster complex for the detection of tumor necrosis factor-alpha based on immunoassay.

Tumor necrosis factor-alpha, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated monocytes and macrophages. Measuring TNF-α levels serves as a valuable indicator for tracking several diseases and pathological states. Gold nanotechnology has been identified as a highly effective catalyst with unique properties for measuring inflammatory cytokines. This study aimed to synthesize gold nanoclusters (AuNCs) and the AuNCs-streptavidin system, along with their characterizations and spherical morphology. The detection of TNF-α antigen with AuNCs was determined, and a new immunoassay-based AuNCs analytical platform was studied. In this study, it was demonstrated that the synthesized AuNCs and AuNCs-streptavidin showed a bright-yellow appearance with absorption peaks at A600 and A610 nm, respectively. The approximately spherical shape was observed by TEM analysis. The AuNCs demonstrated a sensitivity limit for the detection of the TNF-α antigen, with a linear dose-dependent detection range of less than 1.25 ng/mL. The products of the band sizes and band intensities were proportional to the amount of TNF-α in the range of ∼80 kDa, ∼55 kDa, and âˆ¼ 25 kDa in western blot analysis. The TNF-α in cell lysate was successfully detected using an immunoassay after the activation of RAW264.7 cells with lipopolysaccharide (LPS). This assay may serve as a viable alternative for TNF-α detection with high speed, sensitivity, and qualities, ensuring its broad applications.

Ultrasensitive fluorescence microRNA biosensor by coupling hybridization-initiated exonuclease I protection and tyramine signal amplification.

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I (Exo I) protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

An artificial nickel chlorinase based on the biotin-streptavidin technology.

Herein, we report on an artificial nickel chlorinase (ANCase) resulting from anchoring a biotinylated nickel-based cofactor within streptavidin (Sav). The resulting ANCase was efficient for the chlorination of diverse C(sp3)-H bonds. Guided by the X-ray analysis of the ANCase, the activity of the artificial chlorinase could be significantly improved. This approach opens interesting perspectives for late-stage functionalization of organic intermediates as it complements biocatalytic chlorination strategies.

Chemiluminescent plate assay of microRNA-155 coupled with catalytic hairpin assembly with oligonucleotide release (CHAOR).

A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity. The detection limit of microRNA-155 assay was 0.4 pM. The coefficient of variation of the chemiluminescent signal, formed upon the heterogeneous determination of miRNA-155, was less than 12 % within the working range. The efficiency of CHAOR as an amplification method was similar to that of traditional CHA, as miRNA-155 assays based on CHAOR and CHA had similar analytical parameters. In addition, the proposed assay was highly specific. Contrary to traditional CHA, CHAOR, one of whose products is a single-stranded oligonucleotide, can be used in analytical methods based on cascade amplification.

IMBAS-MS Discovers Organ-Specific HLA Peptide Patterns in Plasma.

Distinction of non-self from self is the major task of the immune system. Immunopeptidomics studies the peptide repertoire presented by the human leukocyte antigen (HLA) protein, usually on tissues. However, HLA peptides are also bound to plasma soluble HLA (sHLA), but little is known about their origin and potential for biomarker discovery in this readily available biofluid. Currently, immunopeptidomics is hampered by complex workflows and limited sensitivity, typically requiring several mL of plasma. Here, we take advantage of recent improvements in the throughput and sensitivity of mass spectrometry (MS)-based proteomics to develop a highly sensitive, automated, and economical workflow for HLA peptide analysis, termed Immunopeptidomics by Biotinylated Antibodies and Streptavidin (IMBAS). IMBAS-MS quantifies more than 5000 HLA class I peptides from only 200 µl of plasma, in just 30 min. Our technology revealed that the plasma immunopeptidome of healthy donors is remarkably stable throughout the year and strongly correlated between individuals with overlapping HLA types. Immunopeptides originating from diverse tissues, including the brain, are proportionately represented. We conclude that sHLAs are a promising avenue for immunology and potentially for precision oncology.

Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies.

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

Hybridization chain reaction assisted multicolor immunosensor for sensitively detection of human chorionic gonadotropin.

The level of human chorionic gonadotropin (HCG) is an important indicator for early pregnancy, pregnancy-related diseases trophoblastic diseases and even cancer diagnosis. Therefore, sensitive detection of HCG has crucial significance in clinical, especially in gynaecology and obstetrics. Herein, a hybridization chain reaction (HCR) assisted multicolor immunosensor have been developed for HCG analysis. The proposed method introduced HCR after the immunoreaction between antibody and HCG protein, and produced long double strand DNA (dsDNA) that contain biotin sites. The streptavidin-horseradish peroxidase was linked on the dsDNA by the interaction between biotin and streptavidin, and can further mediated gold nanobipyramids (Au NBPs) etching. The localized surface plasmon resonance absorption peaks of Au NBPs blue shift and accompanied a vivid color change after etching effect. Based on this color change, HCG could be qualitative and semi-quantitative detected. Because of the introduction of HCR and enzyme amplification technique, the proposed method exhibited high sensitivity with a linear range of 0.1-2000 pg/mL and limit of detection (LOD) of 0.1 pg/mL. Finally, the proposed immunosensor was used to detect clinical serum samples. The results show there are no significant differences between clinical results and the test results by this method, indicating the practicability of the proposed method.

Development of a semi-automated MHC-associated peptide proteomics (MAPPs) method using streptavidin bead-based immunoaffinity capture and nano LC-MS/MS to support immunogenicity risk assessment in drug development.

Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.

Construction of a Quantum-Dot-Based FRET Nanosensor through Direct Encoding of Streptavidin-Binding RNA Aptamers for N6-Methyladenosine Demethylase Detection.

N6-Methyladenosine (m6A) demethylases can catalyze the removal of the methyl modification on m6A, and it is closely associated with the occurrence, proliferation, differentiation, and metastasis of malignancies. The m6A demethylases (e.g., fat mass and obesity-associated protein (FTO)) may act as a cancer biomarker and are crucial for anticancer drug screening and early clinical diagnosis. Herein, we demonstrate the construction of a quantum-dot-based Förster resonance energy-transfer (FRET) nanosensor through direct encoding of streptavidin-binding RNA aptamers (SA aptamers) for m6A demethylase detection. This nanosensor employs multiple Cy5-molecule-labeled SA aptamers as the building materials to construct the 605QD-RNA-Cy5 nanoassembly, and it exploits the hinder effect of m6A upon elongation and ligation reactions to distinguish m6A-containing RNA probes from demethylated RNA probes. When m6A demethylase is present, the m6A-containing RNA probes are demethylated to generate the demethylated RNA probes, initiating strand extension and ligation reactions to yield a complete transcription template for SA aptamers. Subsequently, a T7-assisted cascade transcription amplification reaction is activated to transcribe abundant SA aptamers with the incorporation of multiple Cy5 fluorophores. The Cy5-incorporated SA aptamers can self-assembly onto the streptavidin-coated 605QD surface to obtain the 605QD-SA aptamer-Cy5 nanoassemblies, resulting in the generation of distinct FRET signals. This nanosensor exhibits ultrahigh sensitivity and excellent specificity, and it can detect endogenous FTO at the single-cell level. Furthermore, this nanosensor can precisely measure enzyme kinetic parameters, screen m6A demethylase inhibitors, and differentiate the FTO expression between breast cancer patients and healthy individual tissues, offering a versatile platform for clinical diagnostic and drug discovery.