Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

Identifying challenges and opportunities for histone peptides in protein engineering platforms

Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.

Directed Evolution of Aptamer Discovery Technologies

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

A theoretical framework for proteome-scale single-molecule protein identification using multi-affinity protein binding reagents

The proteome is perhaps the most dynamic and valuable source of functional biological insight. Current proteomic techniques are limited in their sensitivity and throughput. A typical single experiment measures no more than 8% of the human proteome from blood or 35% from cells and tissues. Here, we introduce a theoretical framework for a fundamentally different approach to proteomics that we call Protein Identification by Short-epitope Mapping (PrISM). PrISM utilizes multi-affinity reagents to target short linear epitopes with both a high affinity and low specificity. PrISM further employs a novel protein decoding algorithm that considers the stochasticity expected for single-molecule binding. In simulations, PrISM is able to identify more than 98% of proteins across the proteomes of a wide range of organisms. PrISM is robust to potential experimental confounders including false negative detection events and noise. Simulations of the approach with a chip containing 10 billion protein molecules show a dynamic range of 11.5 and 9.5 orders of magnitude for blood plasma and HeLa cells, respectively. If implemented experimentally, PrISM stands to rapidly quantify over 90% of the human proteome in a single experiment, potentially revolutionizing proteomics research.

Advances in Molecularly Imprinted Polymers Based Affinity Sensors (Review)

Recent challenges in biomedical diagnostics show that the development of rapid affinity sensors is very important issue. Therefore, in this review we are aiming to outline the most important directions of affinity sensors where polymer-based semiconducting materials are applied. Progress in formation and development of such materials is overviewed and discussed. Some applicability aspects of conducting polymers in the design of affinity sensors are presented. The main attention is focused on bioanalytical application of conducting polymers such as polypyrrole, polyaniline, polythiophene and poly(3,4-ethylenedioxythiophene) ortho-phenylenediamine. In addition, some other polymers and inorganic materials that are suitable for molecular imprinting technology are also overviewed. Polymerization techniques, which are the most suitable for the development of composite structures suitable for affinity sensors are presented. Analytical signal transduction methods applied in affinity sensors based on polymer-based semiconducting materials are discussed. In this review the most attention is focused on the development and application of molecularly imprinted polymer-based structures, which can replace antibodies, receptors, and many others expensive affinity reagents. The applicability of electrochromic polymers in affinity sensor design is envisaged. Sufficient biocompatibility of some conducting polymers enables to apply them as “stealth coatings” in the future implantable affinity-sensors. Some new perspectives and trends in analytical application of polymer-based semiconducting materials are highlighted.

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

Mapping the binding site topology of amyloid protein aggregates using multivalent ligands

Multivalent ligands offer a powerful approach to obtain high affinity reagents to bind the aggregates that form in neurodegenerative disease. Selectivity for different proteins was achieved by using different linkers to connect the head groups.