Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors.

BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.

A dual-tag microarray platform for high-performance nucleic acid and protein analyses.

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.

Self-assembly of proximity probes for flexible and modular proximity ligation assays.

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

In vitro analysis of DNA-protein interactions by proximity ligation.

Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.

Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development.

During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor beta (PDGFRbeta) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.

Sensitive protein detection via triple-binder proximity ligation assays.

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.

Proximity ligation: a specific and versatile tool for the proteomic era.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

A sensitive proximity ligation assay for active PSA.

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.

Detection of individual microbial pathogens by proximity ligation.

BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity. METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents. RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium. CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes.

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.

Cytokine detection by antibody-based proximity ligation.

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.

Padlock and proximity probes for in situ and array-based analyses: tools for the post-genomic era.

Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.