Comparison of the specificity and affinity of surface immobilised Affimer binders using the quartz crystal microbalance.

To enable multiplexed protein analysis through the use of microarrays, reliable molecules capable of specifically binding to a protein of interest with high affinity are required. Further, this specificity and affinity must be retained upon immobilization to the microarray surface. This study investigates the performance of surface bound Affimer proteins, comparing the affinity and specificity of different binders for closely related immunoglobulin molecules using the quartz crystal microbalance with dissipation monitoring (QCM-D). It is demonstrated that the surface bound Affimer proteins are highly specific, differentiating between their target IgG and other closely related IgG subclasses. The binding affinities of the protein aptamers for their target IgG molecules are determined to be in the nanomolar range, comparable to typical antibody-antigen binding affinities. While measurements herein are done using QCM-D, the high specificity and binding affinities of the surface bound Affimer proteins opens applications in a range of microarray biosensors.

Sensitive affimer and antibody based impedimetric label-free assays for C-reactive protein.

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. Levels greater than 3 µg/mL serum have hitherto been considered to indicate pathology, but there is increasing interest in assessments between 0.1 and 10 µg/mL, which have been found to correlate with severity of risk for cardiovascular disease. We report herein the generation of both antibody and Affimer based impedance immunoassays for CRP that are substantially more sensitive than clinically utilized immunonephelometry and immunoturbidity assessments. Significant in this study is not only the use of a constrained peptide to detect a clinically important target but also that derived electrochemical impedance assays can be highly sensitive even with probes whose relatively weak (µM) affinities are not amenable to target detection by surface plasmon resonance (SPR). Key to this finding is acknowledging that receptive surfaces of comparatively low initial steric bulk and charge transfer resistance are especially primed to be highly responsive to target binding in electroanalytical assays of this type.

The role of BCL6 in lymphomas and routes to therapy.

BCL6 is a transcription factor that has essential B-cell and T-cell roles in normal antibody responses. It is involved in chromosomal translocations in diffuse large B-cell lymphoma (DBCL; including primary mediastinal B-cell lymphoma) and nodular lymphocyte predominant Hodgkin lymphoma, and is expressed in follicular lymphoma and Burkitt's lymphoma. The neoplastic T-cells of angioimmunoblastic T-cell lymphoma also express BCL6. BCL6 prevents terminal B-cell differentiation largely through repression of PRDM1. In the "cell of origin" classification of DLBCL BCL6 is associated with the germinal centre subtype, which carries a good response to modern treatments. More recently, specific BCL6 antagonists, including small molecule inhibitors, have been developed. These antagonists have demonstrated that DLBCL cells, in which BCL6 is transcriptionally active, are dependent on this gene for survival. BCL6 antagonists are active against primary DLBCL and may find future application in the treatment of lymphomas.

Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

BACKGROUND: Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. RESULTS: Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long) intracellular domain of membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the mu2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. CONCLUSIONS: Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.

Label-free sub-picomolar protein detection with field-effect transistors.

Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing.

Peptide aptamers in label-free protein detection: 2. Chemical optimization and detection of distinct protein isoforms.

The early detection and diagnosis of cancer lies central to successful treatment and improved patient outcome. Current techniques are limited by the nature of the biological receptor and the assays available. This paper reports the use of novel biological probes, peptide aptamers, in detecting cyclin-dependent protein kinases (CDKs) whose activity is important in proliferating and cancerous cells. We describe, specifically, the optimization of an orientated peptide aptamer surface and its utilization in establishing a highly specific, low-nanomolar sensitive, detection protocol for the active form of CDK2. In comparing target binding affinity of two different aptamers (pep6 and pep9), both constructed through the insertion of peptide sequences into the surface of a scaffold protein, one was observed to be consistently more effective. Significantly, the pep9 aptamers were able to detect subtle changes in the conformation of CDK2 associated with activation of its catalytic activity that may be caused by the phosphorylation of a single amino acid (threonine 160). A typical response toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of the active form of CDK2 in the concentration range from 2 to 20 nM. Although antibodies are occasionally able to recognize conformations in their targets, this is the first time that a nonantibody protein probe has been used to detect an active protein isoform. Because peptide aptamers are usually raised against full-length proteins, this raises the possibility that peptide aptamers will be able to extend the repertoire of probes that recognize protein conformations, post-translational modifications (PTMs), or conformations stabilized by PTMs.

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors.

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80 nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22 mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection.

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors.

Design and validation of a neutral protein scaffold for the presentation of peptide aptamers.

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.

Non-antibody protein-based biosensors.

Biosensors that depend on a physical or chemical measurement can be adversely affected by non-specific interactions. For example, a biosensor designed to measure specifically the levels of a rare analyte can give false positive results if there is even a small amount of interaction with a highly abundant but irrelevant molecule. To overcome this limitation, the biosensor community has frequently turned to antibody molecules as recognition elements because they are renowned for their exquisite specificity. Unfortunately antibodies can often fail when immobilised on inorganic surfaces, and alternative biological recognition elements are needed. This article reviews the available non-antibody-binding proteins that have been successfully used in electrical and micro-mechanical biosensor platforms.

The ansamycin antibiotic, rifamycin SV, inhibits BCL6 transcriptional repression and forms a complex with the BCL6-BTB/POZ domain.

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ∼40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor.

Peptide aptamer microarrays: bridging the bio-detector interface.

In the near future, personalised medicine and phase-0 trials will require that clinical practitioners move from the "one biomarker per disease" paradigm to the use of molecular signatures of disease for diagnosis and the prediction of a patient's response to treatment. These signatures will be composed of biomarkers specific to the disease, and will include over-expression of normal protein from a gene that does not carry a mutation; loss of expression of an essential protein; expression of a protein from a mutant gene; and metabolites whose levels are altered in disease. Surrogates for protein expression, such as alterations in the messenger RNA that encode for them, have already proved their value. The next challenge, then, in clinical biosensing is to enable the multiplexed detection of protein biomarkers, and perhaps the multiplexed detection of mixed biomarkers (metabolites, RNA and proteins) all in a single test. Given the plethora of available antibodies specific for biomarkers, why is this not already happening? We believe that the limitation lies in the nature of the antibody molecule itself, and especially the fact that antibodies have evolved to function in solution, while most diagnostic tests take place at a surface. We have accordingly turned to the design of alternative antibodies, and have identified a protein that appears to be unusually stable on surfaces. The new, non-antibody, scaffold protein is derived from human Stefin A, a natural inhibitor of the cathepsin family of proteases. We have engineered this protein so that it lacks natural binding partners, and introduced a series of new binding surfaces through randomisation or directed replacement of the surfaces used by Stefin A to bind to cathepsins. Our new probes show exquisite specificity and binding affinities comparable to antibodies, and can be used to probe biology in intact cells. More importantly, together and in collaboration with other groups in Chemistry or Engineering Departments, we have shown that these designer proteins can be used in optical detection of labelled target proteins from whole cell lysates in a highly multiplexed microarray format, as well as in label-free detection of unlabelled proteins using surface plasmon resonance, QCM, microcantilevers and using electrochemical assays on gold electrodes. We believe that the combination of optimised surface chemistry, robust and combinatorial designer biological probes and novel, robust and sensitive detection technologies will enable, in the near future, the introduction of multiplexed biomarker detection in the clinical setting, most likely in cancer where multiple biomarkers are known, but probes are still lacking.

Optimisation of a multivalent Strep tag for protein detection.

The Strep tag is a peptide sequence that is able to mimic biotin's ability to bind to streptavidin. Sequences of Strep tags from 0 to 5 have been appended to the N-terminus of a model protein, the Stefin A Quadruple Mutant (SQM) peptide aptamer scaffold, and the recombinant fusion proteins expressed. The affinities of the proteins for streptavidin have been assessed as a function of the number of tags inserted using a variety of labelled and label-free bioanalytical and surface based methods (Western blots, microarray assays and surface plasmon resonance spectroscopy). The binding affinity increases with the number of tags across all assays, reaching nanomolar levels with 5 inserts, an observation assigned to a progressive increase in the probability of a binding interaction occurring. In addition a novel interfacial FRET based assay has been developed for generic Strep tag interactions, which utilises a conventional microarray scanner and bypasses the requirement for expensive lifetime imaging equipment. By labelling both the tagged StrepX-SQM(2) and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences.

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.

Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays.

BACKGROUND: The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. RESULTS: We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. CONCLUSION: The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

Surface-immobilized peptide aptamers as probe molecules for protein detection.

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.

Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold.

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.

Molecular analysis of survivin isoforms: evidence that alternatively spliced variants do not play a role in mitosis.

Survivin is a protein with proposed roles in cell division and apoptosis. Transcripts encoding splice variants of human survivin have been described and their expression correlated with cancer progression. As survivin forms homodimers in vitro, it has been suggested that these isoforms could interfere with wild type function by forming heterodimers. Here we show that survivin-2beta and survivin-deltaEx3 can interact with wild type survivin but have reduced affinity for the partner protein of survivin, borealin, and thus do not localize with the chromosomal passenger complex in vivo. Furthermore, we demonstrate that overexpression of survivin-2beta-green fluorescent protein (GFP) or survivin-deltaEx3-GFP does not impede cell cycle progression. We also report that wild type survivin, but not survivin-2beta-GFP or survivin-deltaEx3-GFP, can rescue cell proliferation inhibited by small interfering RNA-mediated survivin depletion. These data suggest that, despite their ability to interact with wild type survivin, neither of these isoforms acts as its competitor during mitosis nor has an essential function.