Streptavidin-coated Beads Research Articles

A rapid point-of-care population-scale dipstick assay to identify and differentiate SARS-CoV-2 variants in COVID-19-positive patients.

Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion-deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences from 30 distinct SARS-CoV-2 variants, focusing on the unique nucleic acid signatures in the spike gene specific to the Delta and Omicron variants. Using these variant-specific sequences, we synthesized a range of oligonucleotides and optimized a multiplex PCR (mPCR) assay capable of accurately identifying and differentiating between the Delta and Omicron variants. Building on this mPCR assay, we developed a dipstick format by incorporating a tag linker sequence at the 5' end of the forward primer and adding biotin to the 3' end of the oligonucleotides, enhancing the assay's usability and accessibility. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.

In Vitro Selection and Characterization of a Light-up DNA Aptamer for Thiazole Orange.

A new DNA aptamer that binds to the target Thiazole Orange-biotin (TO1-biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin-coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off-rate binding. Using next-generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three-tiered G-quadruplex motif for these families. This indicates that the TO1-biotin DNA aptamer identified here uses a similar ligand-binding topology to the original Mango RNA aptamer.

Chrom-seq identifies RNAs at chromatin marks.

Chromatin marks are associated with transcriptional regulatory activities. However, very few lncRNAs have been characterized with the role in regulating epigenetic marks, largely due to the technical difficulty in identifying chromatin-associating RNA. Current methods are largely limited by the availability of ChIP-grade antibody and the crosslinking, which generates high noise. Here, we developed a method termed Chrom-seq to efficiently capture RNAs associated with various chromatin marks in living cells. Chrom-seq jointly applies highly specific chromatin mark reader with APEX2, which catalyzes the oxidation of biotin-aniline to label the adjacent RNAs for isolation by streptavidin-coated beads. Using the readers of mCBX7/dPC, mCBX1, and mTAF3, we detected RNA species significantly associated with H3K27me3, H3K9me3, and H3K4me3, respectively. We demonstrated that Chrom-seq outperformed other equivalent methods in terms of sensitivity, efficiency, and cost of practice. It provides an antibody-free approach to systematically map RNAs at chromatin marks with potential regulatory roles in epigenetic events.

Improvements of magnetic nanoparticle assays for SARS-CoV-2 detection using a mimic virus approach

Immunoassays with magnetic nanoparticles (MNPs) as markers are a promising approach for the fast and sensitive virus detection. Upon binding of antibody-functionalized MNP on virus proteins, the hydrodynamic diameter increases and a change in the Brownian relaxation time can be measured. In this study, we detect the whole SARS-CoV-2 by mimicking it with streptavidin-coated polystyrene beads with biotinylated spike proteins. Changes of the MNP dynamics are measured by alternating current susceptometry and magnetic particle spectroscopy. Due to the multiple binding sites of MNP and virus, crosslinking enlarges the change of the hydrodynamic diameter. In order to improve the sensitivity and the limit of detection of the assay, the ratio of the virus to the MNP amount RMV/MNP is investigated in detail. High RMV/MNP ratios lead to a saturation of the MNPs with viruses, so that the cluster size and therefore the sensitivity decrease again. Additionally, it is found that the smallest virus concentrations can be detected for small MNP concentrations. It is also shown that the RMV/MNP range that can be used for an unambiguous detection of viruses depends on the virus/MNP concentration; it shifts with increasing MNP concentration to smaller RMV/MNP values. For very small virus concentrations, an increase of the Brownian relaxation time is detected implying a decrease of the hydrodynamic diameter. Furthermore, the optimal antibody concentration for MNP functionalization was determined. It is also found that a washing process with a centrifuge improves the sensitivity by reliably removing unbound antibodies and eliminating small MNPs with improper functionalization.

Membrane Tubulation with a Biomembrane Force Probe.

Tubulation is a common cellular process involving the formation of membrane tubes ranging from 50 nm to 1 µm in diameter. These tubes facilitate intercompartmental connections, material transport within cells and content exchange between cells. The high curvature of these tubes makes them specific targets for proteins that sense local geometry. In vitro, similar tubes have been created by pulling on the membranes of giant unilamellar vesicles. Optical tweezers and micromanipulation are typically used in these experiments, involving the manipulation of a GUV with a micropipette and a streptavidin-coated bead trapped in optical tweezers. The interaction forms streptavidin/biotin bonds, leading to tube formation. Here, we propose a cost-effective alternative using only micromanipulation techniques, replacing optical tweezers with a Biomembrane Force Probe (BFP). The BFP, employing a biotinylated erythrocyte as a nanospring, allows for the controlled measurement of forces ranging from 1 pN to 1 nN. The BFP has been widely used to study molecular interactions in cellular processes, extending beyond its original purpose. We outline the experimental setup, tube formation and characterization of tube dimensions and energetics, and discuss the advantages and limitations of this approach in studying membrane tubulation.

Highly efficient identification of nucleocytoplasmic O-glycosylation by the TurboID-based proximity labeling method in living cells.

Glycosylation is a ubiquitous posttranslational modification and plays an important role in many processes, such as protein stability, folding, processing, and trafficking. Among glycosylation types, O-glycosylation is difficult to analyze due to the complex glycan composition, low abundance and lack of glycosidases to remove the O-glycans. Many methods have been applied to analyze the O-glycosylation of membrane glycoproteins and secreted glycoproteins since the synthesis of O-glycosylation occurred in the Golgi apparatus. In recent years, some O-glycosylation has been reported in the nucleus. In this work, we present a proximity labeling strategy based on TurboID by combining core 1 β1-3 galactosyltransferase (C1GalT1), which has been reported in the nucleus, to characterize nucleocytoplasmic O-glycosylation in living HeLa cells. The O-glycosylated protein C1GalT1 was biotinylated by the proximity labeling method in living HeLa cells overexpressing C1GalT1 fused by TurboID and enriched by streptavidin-coated beads. Following digestion with trypsin and mass spectrometry analysis, 68 high-confidence and 298 putative O-glycosylated sites were identified on 366 peptides mapped to 267 proteins. These results indicated that the proximity labeling method is a highly efficient technique to identify O-glycosylation. Furthermore, the finding of abundant O-glycosylation from nucleocytoplasmic proteins indicates a new pathway of O-glycosylation synthesis in cells.

A 3D paper microfluidic device for enzyme-linked assays: Application to DNA analysis.

A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (μEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the μEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the μEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The μEL-PAD achieved a limit of detection (LOD) and quantification<10 genome copies/μL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.

Coupling hCG-based protease sensors with a commercial pregnancy test strip for simple analyses of protease activities

Proteases play an essential role in many cellular processes, and consequently, abnormalities in their activities are related to various diseases. Methods have been developed to measure the activity of these enzymes, but most involve sophisticated instruments or complicated procedures, which hampers the development of a point-of-care test (POCT). Here, we propose a strategy for developing simple and sensitive methods to analyze protease activity using commercial pregnancy test strips that detect human chorionic gonadotropin (hCG). hCG was engineered to have site-specific conjugated biotin and a peptide sequence, which can be cleaved by a target protease, between hCG and biotin. hCG protein was immobilized on streptavidin-coated beads, resulting in a protease sensor. The hCG-immobilized beads were too large to flow through the membrane of the hCG test strip and yielded only one band in the control line. When the peptide linker was hydrolyzed by the target protease, hCG was released from the beads, and the signal appeared in both the control and test lines. Three protease sensors for matrix metalloproteinase-2, caspase-3, and thrombin were constructed by replacing the protease-cleavable peptide linker. The combination of the protease sensors and a commercial pregnancy strip enabled the specific detection of each protease in the picomolar range, with a 30-min incubation of the hCG-immobilized beads and samples. The modular design of the protease sensor and simple assay procedure will facilitate the development of POCTs for various protease disease markers.

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed. To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Micheliolide Exerts Therapeutic Effect in Myeloproliferative Neoplasms through Binding to STAT3 and STAT5 Proteins and Inhibiting Their Activities

The JAK1/2 inhibitor ruxolitinib is a cornerstone of management for some subsets of BCR-ABL negative myeloproliferative neoplasms (MPN). While many patients benefit with reduced splenomegaly and symptoms, some patients respond suboptimally to ruxolitinib, and even patients with a good initial response often develop resistance to ruxolitinib after several years of therapy. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, in the JAK2V617F positive MPN entities. We firstly evaluated the effect of MCL treatment on cell growth and survival in human MPN cell lines expressing the JAK2V617F mutation. MCL alone reduced cell viability and induced apoptosis of UKE1 and SET2 cells in a dose-dependent manner, and obviously improved the effect of ruxolitinib as monotherapy. Importantly, treatment with MCL also induced significant apoptosis of UKE1 cells which were selected for resistance against ruxolitinib. We further explored the therapeutic potential of MCL in vivo using a Jak2V617F knock-in (Jak2VF) mouse model, and found that DMAMCL, an orally available derivative of MCL, significantly increased ruxolotinib efficacy in Jak2VF mice, manifested as amplified hematological and spleen responses. What's more, DMAMCL, whereas not ruxolitinib, contributed to reduce the in vivo Jak2V617F mutant allele burden reflected by the percentage of mutant hematopoietic cells in peripheral blood and bone marrow. To determine the molecular mechanisms by which MCL exerts efficacy in MPN, we firstly performed western blot (WB) anlyses to evaluate the effect of MCL on hyper-activated JAK-STAT signaling, which is the cornerstone of MPN pathogenesis. We observed a dose-dependent inhibition of STAT3 and STAT5 phosphorylation caused by MCL treatment in UKE1 and SET2 cells (Figure 1A), and as expected, combination MCL with ruxolitinib provided an enhanced inhibitory effect. By next performing the affinity pull-down experiment, we confirmed that MCL suppressed the activation of STAT3 and STAT5 mainly through directly binding to the proteins (Figure 1B). We further conducted RNA-sequencing analysis in UKE1 cells treated with MCL and/or ruxolitinib to gain insights into mechanisms involved in MCL effect. Gene set enrichment analysis (GSEA) showed that genes related to TNF-α-NF-κB signaling and inflammatory response pathway were significantly downregulated upon treatment with MCL, that were verified by WB analyses and real-time quantitative PCR. Additionally, GSEA also revealed significant upregulation of genes related to reactive oxygen species (ROS) and unfolded protein response (UPR) pathway, respectively. Both flow cytometry and fluorescent microscopy imaging confirmed the ROS accumulation by MCL treatment. We also determined the activation of PERK branch of UPR pathway caused by MCL, as assessed by increased phosphorylation of eIF2α, and upregulation of pro-apoptotic genes, including ATF4, CHOP and GADD34. Finally, by addition of antioxidant agent, the activation of UPR pathway and increase of apoptosis in UKE1 cells induced by MCL were significantly rescued, indicating that MCL exerts cytotoxic effects mainly through triggering the oxidative stress in MPN cells. In summary, our results demonstrate that MCL is an effective agent with multiple biological activities in MPN, especially inhibition of STAT3/5 activity via directly binding to the proteins, and may be a promising drug combined with ruxolitinib to benefit patients who have suboptimal response or acquire resistance to ruxolitinib. Figure 1. MCL inhibits STAT3 and STAT5 phosphorylation through binding to the proteins directly. (A) Western blot analyses are shown for phosphorylated-STAT3 (p-STAT3), STAT3, phosphorylated-STAT5 (p-STAT5) and STAT5 levels in total cell extracts from UKE1 and SET2 cells treated with MCL (5, 10, 20μM) for 3 hours. (B) The biotin-conjugated MCL (biotin-MCL) and its inactive analogue S-MCL (biotin-S-MCL) were prepared as the positive- and negative- probe respectively. UKE1 and SET2 cell lysates were incubated with 20μM biotin-MCL or biotin-S-MCL for 1 hour at room temperature, then the mixtures were separately pulled down with streptavidin-coated agarose beads, and STAT3/STAT5 were detected with WB analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anwulignan Suppresses Myeloma Growth By Pyroptosis

Purpose Multiple myeloma resistance to apoptosis and antitumor immunity contribute to poor therapeutic responses. Pyroptosis, an inflammatory programmed cell death distinct from apoptosis, has potential to be clinically harnessed and to address these problems. Chinese traditional medicine has advanced the care of patients; however, detailed knowledge of the medicine is lacking. Anwulignan is a monomer compound of the Chinese traditional medicine Schisandra sphenanthera lignans. The goal of this study is to determine whether anwulignan could suppress myeloma growth by activating pyroptosis, enhancing anti-tumour immunity. Methods 1. IC50 values of four MM cell lines to anwulignan were detected to determine the effect of anwulignan on myeloma cells. To determine whether pyroptosis was involved during anwulignan treatment, markers of pyroptotic cell death, cleavage of gasdermin (GSDM), caspase 3 and release of HMGB1, IL-18, IL-1β were detected. 2. High-throughput mRNA sequencing was used to screen out the differential genes after anwulignan treatment. Differential genes enrichment analysis based on GO (Gene Ontology Resource), KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to clarify the signaling pathways and biological processes affected by anwulignan. 3. To ascertain the functional contribution of the immune system in anwulignan therapeutic efficacy, we compared tumor responses in immunodeficient mice and immunocompetent mice. Orthotopic MM xenograft models were established in NOD-Prkdcscid Il2rgtm1/Bcgen (NSG) mice with luciferase labelled LP-1 cells. Orthotopic MM allografts models were established in C57Bl/KaLwRij mice with luciferase labelled 5TGM1 cells. Tumor burden was assessed by serial bioluminescence imaging. 4. To determine whether the antitumor immune responses during anwulignan treatment were dependent on mediators of pyroptosis, tumor-infiltrating T lymphocyte populations from control or GSDMD-KO tumors treated with anwulignan were detected. 5. To identify the mechanism and targets underlying the inhibitory anwulignan activity, we performed a high-throughput proteomics platform to screen the potential targets of anwulignan. We synthesized biotinylated anwulignan to lable anwulignan. MM.1S cell lysates were incubated with biotin-anwulignan or free biotin, and the mixtures were precipitated with streptavidin-coated agarose beads, followed by gel electrophoresis and silver staining. Only one band was clearly precipitated by biotin-anwulignan but not by free biotin. The differential band was prepared for LC-MS/MS analysis. Cluster analysis was used to screen out the preferred binding partners of anwulignan. Results 1. CCK8 assays showed that the IC50 values were found to be 38.29μM, 51.09μM, 70.61μM and 87.16μM at 72 h for LP-1, ARH-77, U266, and MM.1S cells, respectively. In addition to increased cleavage of GSDMD and caspase 3, anwulignan treatment promoted release of HMGB1 and IL-18, indicating that pyroptosis occurred in anwulignan-treated myeloma cells. 2. RNA-seq results revealed that the expression of pyroptosis genes increased significantly in anwulignan treatment group, suggesting that anwulignan induces pyroptotic cell death. 3. Tumors in either immunodeficient mice (Figure 1A) or immunocompetent mice showed robust tumor regression following anwulignan treatment. However, anwulignan induced prolonged tumor regressions in C57Bl/KaLwRij mice, with tumors taking an average of 106.5 days to regrow to 10s pscr compared with short-term regressions averaging 36.2 days in NSG mice. Moreover, we observed more extended overall survival of anwulignan-treated C57Bl/KaLwRij mice compared with NSG mice. These data suggest that an intact immune system significantly contributes to the therapeutic efficacy of anwulignan. 4. Higher levels of CD8+ T cells were detected in control tumors in comparison with the GSDMD-KO counterparts 5. High-throughput proteomics platform detected a series of candidate proteins that may bind to anwulignan (Figure 1B). Cluster analysis was performed using STRING software and showed that anwulignan preferably binds to pyroptosis proteins. Conclusion These data implicate the natural product anwulignan-induced pyroptosis in anti-myeloma immune responses and highlight new therapeutic strategies for MM. Our data suggest that pyroptosis may be an indispensable mediator of immune-driven therapeutic response in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.

Magnetic beads for the evaluation of drug release from biotinylated polymeric micelles in biological media.

To improve the reliability of in vitro release studies of drug delivery systems, we developed a novel in vitro method for the evaluation of drug release from polymeric micelles in complex biological media. Polymeric micelles based on poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) of which 10% of the chains was functionalized with biotin at the p(HPMAm) terminus were prepared using a solvent extraction method. The size of the micelles when loaded with a hydrophobic agent, namely paclitaxel (a clinically used cytostatic drug) or curcumin (a compound with multiple pharmacological activities), was around 65nm. The biotin decoration allowed the binding of the micelles to streptavidin-coated magnetic beads which occurred within 10min and reached a binding efficiency of 90±6%. Drug release in different media was studied after the magnetic separation of micelles bound to the streptavidin-coated beads, by determination of the released drug in the media as well as the retained drug in the micellar fraction bound to the beads. The in vitro release of paclitaxel and curcumin at 37°C in PBS, PBS containing 2% v/v Tween 80, PBS containing 4.5% w/v bovine serum albumin, mouse plasma, and whole mouse blood was highly medium-dependent. In all media studied, paclitaxel showed superior micellar retention compared to curcumin. Importantly, the presence of serum proteins accelerated the release of both paclitaxel and curcumin. The results presented in this study show great potential for predicting drug release from nanomedicines in biological media which in turn is crucial for their further pharmaceutical development.

Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks

Herpes simplex virus type 1 (HSV‐1) is a ubiquitous pathogen that replicates the 152kbp viral genome within the nucleus of host cells. Replication of the HSV‐1 genome is catalyzed by viral replication machinery consisting of a DNA polymerase (UL30), processivity factor (UL42), helicase‐primase complex, origin binding protein (UL9), and a single stranded DNA binding protein. Despite encoding a viral DNA polymerase processivity factor, we found that host Proliferating Cell Nuclear Antigen (PCNA) interacts with HSV‐1 DNA at replication forks and associates with viral DNA in a replication‐dependent manner. PCNA is a homotrimer that provides processivity to cellular DNA polymerases and selectively recruits DNA damage response and DNA repair factors to cellular replication forks. We therefore hypothesized that PCNA associates at viral replication forks to promote HSV‐1 replication while also tethering cellular DNA repair proteins to replicating viral DNA. To test this, we used two models to identify how associated factors change as a function of PCNA: a known PCNA inhibitor, PCNA‐I1, and a cell line that was engineered to inducibly express a shRNA targeting PCNA. We performed a technique adapted from isolation of proteins on nascent DNA (iPOND) to isolate HSV‐1 DNA from infected cells and identified associated viral and cellular proteins. Cells were infected and replicating viral DNA was selectively labeled with EdC. Labeled DNA was specifically and irreversibly tagged via the covalent attachment of biotin azide via click reaction. Biotin‐tagged DNA was purified on streptavidin‐coated beads and associated proteins were eluted and identified by mass spectrometry. As a complimentary approach, we also performed immunofluorescence (IF) to confirm iPOND data. We found that PCNA is associated with viral DNA despite treatment with PCNA‐I1, an observation that is consistent with IF imaging data. Viral replication proteins UL30, UL42, and UL9 decreased in the presence of PCNA‐I1. DNA damage response proteins such as mismatch repair proteins and RECQL both decreased up to 10‐fold. Of note, however, proteins that make up the MRN complex, a class of proteins that functions in DNA break repair, restart of stalled replication forks, and viral DNA infection, increased in response to PCNA‐I1 treatment. Given this data, PCNA may be involved in tethering viral replication proteins, cellular DNA repair proteins, and/or virion assembly proteins to viral replication forks.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

Identification of MPK4 kinase interactome using TurboID proximity labeling proteomics in Arabidopsis thaliana.

TurboID is a new and efficient proximity labeling system that was first developed in living mammalian cells. TurboID is a modified bacterial biotin ligase that can be fused to a bait protein, which can then modify proximal interacting proteins with biotin. Prey proteins subsequently labeled with biotin tags will be pulled down with streptavidin-coated beads and identified by mass spectrometry-based proteomics. TurboID has been recently applied to living plant cells and provided promising results in identification of interacting proteins. Mitogen-activated protein kinase 4 (MPK4) is important for plant growth, development, and defense; however, the molecular mechanisms underlying the range of MPK4 functions are not completely known. Here we use modern proteomics together with the TurboID in a proof-of-concept study to profile the MPK4 interactome and uncover the functions of MPK4 in plant signaling cascades.

Capture the Tag: Mitigation of Biotin Interference in ELISA and Automated Immunoassays by Pre-Conjugating Biotinylated Antibodies to the Streptavidin Surface

Abstract Introduction Streptavidin to biotin binding is one of the strongest non-covalent interactions in nature, and is therefore, successfully incorporated into many immunoassays to facilitate antibody capture. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase the result depending on assay format. Currently, biotin-stripping methods capture free biotin in patient specimens by pre-incubation with streptavidin-coated microparticles. However, this approach increases turnaround time, test cost, and testing steps, which increases the chance of error. Our objective was to determine if pre-conjugating biotinylated antibodies to the assay’s streptavidin solid surface before adding patient specimen could mitigate biotin interference in ELISA and automated sandwich immunoassays. Methods We performed this study on 3 different ELISAs (CYFRA-21, NSE, S100B) and one automated assay (thyroglobulin); all are a sandwich immunoassay format. Serum pools were spiked with biotin (25 - 1000 µg/L) or PBS control. Manufacturer protocols were followed in both the ELISAs and automated assay to evaluate baseline concentration-dependent biotin interference. Mitigation of biotin interference by pre-incubation was then evaluated in the ELISAs by adding biotinylated antibody to the streptavidin-coated wells 0, 10, 15, or 60 min before adding biotin- or PBS-spiked serum specimens. For the automated assay, streptavidin-coated beads and biotinylated antibody were removed from the reagent cartridge, mixed, and incubated 4.5 hours. The mixture containing biotin-tagged antibody bound to streptavidin beads was added back to the cartridge and placed on the analyzer to evaluate spiked specimens. Lastly, we compared the pre-incubation method to a standard biotin-stripping protocol in the ELISAs to compare the effectiveness of mitigating biotin interference. Results We observed biotin interference across the three ELISAs, where 400 µg/L biotin spiked in serum pools reduced analyte detection to between 10 – 15% of the total activity using the standard assay format. Our time-course studies showed 84 – 95% recovery of the total activity when the biotinylated antibody was pre-incubated in the streptavidin-coated wells for 1 hour prior to addition of serum specimens, compared to 69 – 99% by a standard biotin stripping protocol. We extended this concept to the automated immunoassay where at 1000 µg/L biotin in the specimen, only 9 ±0.01% of the total analyte was measured by the conventional method. However, pre-mixing biotinylated antibody and streptavidin beads resulted in 97 ±0.01% of the total analyte recovery in the presence of 1000 µg/L biotin. Conclusion We have demonstrated that pre-conjugating the biotin antibody to streptavidin is as effective as biotin-stripping methods to avoid biotin interference in sandwich immunoassays that utilize the biotin-streptavidin system, with the additional benefit of optimizing turnaround times, cost, and labor. A simple change in manufacturer assay design could make immunoassays more robust against biotin interference in patient samples.

Capture of the newly transcribed RNA interactome using click chemistry.

Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.

Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics

Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency, which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intraorganelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

Endogenous anti-streptavidin antibodies causing erroneous laboratory results more common than anticipated

All immunological methods are vulnerable to different kinds of interference. The purpose of this work was to study the cause and frequency of method-dependent interference in the Roche thyroid immunoassays. Serum samples with discordant thyroid function tests (TFT) were selected from samples sent to the Hormone Laboratory, Oslo University Hospital from June 2013 to September 2018. We identified 93 patients with discordant pathological TFT when analysed with the Roche methods and normal results when analysed with alternative methods. 42 of these samples were sent to Roche Diagnostics (Germany) for investigation of the interfering substance. Roche found interference to be caused by the presence of endogenous anti-streptavidin antibodies (ASA) (34 of 42 patients), ruthenium or the idiotype of the ruthenium labelled antibody (3 of 42 patients) and mouse antigens (1 of 42 patients). Method-dependent interference was estimated to affect 0.37% of the patients investigated in our laboratory. Interference due to the presence of endogenous ASA were further explored in other (non-thyroid) immunoassays by comparing analyte levels before and after pre-adsorption of the patients’ sera with streptavidin-coated paramagnetic beads. An underestimation of hormone levels was observed in sandwich immunoassays, while an overestimation was found in competitive assays. Method-dependent interference in Roche thyroid immunoassays is caused mainly by ASA and is not a very rare phenomenon. Misleading results may lead to misdiagnosis and inappropriate medical treatment. The supplier of the assay should be alerted when the available alternative methodology reveals method-dependent errors.