Selection and characterization of DNA aptamers

This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is not exclusively associated with aptamers. Antibodies are the most successful affinity tools used today, but alternative affinity tools such as aptamers, engineered binding proteins and molecular imprinted polymers are emerging as sound alternatives. A comparison of their properties is described in Chapter 1. The strength and specificity of the interaction between an affinity tool and its target molecule is an important feature. Generally, an affinity tool should have a high affinity for its target and should be highly specific in order to be useful for research or commercial purposes. One highly advanced method to characterise the interaction between an affinity tool and its target molecule makes use of a Surface Plasmon Resonance (SPR)-based biosensor. Although SPR is an optical phenomenon, in depth knowledge of the physics behind this phenomenon is not required to operate an SPR-based biosensor. Experiments should be performed in a correct way, and therefore it is important to understand how experimental parameters, such as flow rate, ligand density, surface preparation, and reagent quality either improve or adversely affect data quality. Experimental considerations, as well as methods for proper data analysis are discussed in Chapter 2. Data generated within the framework of the 2011 Global Label-free Interaction Benchmark study serves as a typical example. The ability of aptamers to bind a specific target originates from an intricate interplay between the oligonucleotide sequence and the three dimensional structure that this sequence allows to form. In Chapter 3 this is illustrated by the selection and characterisation of streptavidin-binding aptamers. Five aptamer families were identified, sharing a similar secondary structure. Although slight variations at the actual sequence level are present, two guanines are completely conserved. Using site-specific mutagenesis it was demonstrated that these guanines are essential for streptavidin binding. Binding kinetics and the dissociation constant of each aptamer was determined by SPR and were all within the range of 35-375 nM. Two aptamers can bind one streptavidin tetramer at the same time, as was shown by native mass spectrometry analysis. In addition, the three dimensional structure of the most abundant aptamer was modelled and manually docked to the streptavidin structure, in order to gain more insight in the molecular basis of the interaction. To extend this knowledge even further, crystallisation trails, aiming to obtain a co-crystal structure for the streptavidin-aptamer complex, were performed, and are described in Chapter 4. Unfortunately, these trials did only yield protein crystals, instead of the desired streptavidin-aptamer complex. Therefore, alternative experimental and computational approaches were investigated that could be used to study aptamer-protein interactions. Combining techniques as SPR, small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), and Dynamic light scattering (DLS) could be considered as an alternative to X-ray crystallography. In addition, some of these techniques may provide information on the dynamics of complex formation, whereas crystallography gives a time- and position-averaged image. Besides streptavidin, another protein, SpaC, was subjected to aptamer selection in this thesis. SpaC is a subunit of pili present on the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG and contains a binding domain for human-mucus. Presence of this binding domain is considered an advantage, because it is already designed to interact with other molecules. Successful production and purification of recombinant SpaC protein is described in Chapter 5, as well as the characterisation of DNA oligonucleotides enriched during subsequent selection rounds. Sequence analysis revealed that specific oligonucleotides are indeed enriched. Furthermore, results of pilot SPR experiments indicated that they bind specifically to SpaC, but more detailed experiments are required to unambiguously demonstrate this. The dynamics of aptamer enrichment are poorly understood. To address this issue and to gain a more fundamental insight in the aptamer selection process, a multiplexed high throughput sequencing effort was started, which is described in Chapter 6. In this approach samples of 70 selection rounds, derived from 8 distinct aptamer selection experiments, were barcoded, pooled together and sequenced; over 84 million paired-end reads were obtained and analysed. Samples enriched to bind streptavidin show a decrease in α-diversity across subsequent selection rounds. Interestingly, large differences were found between the composition of fractions enriched by affinity elution and thermal elution. Moreover, a small scale comparison of two clone libraries showed that affinity elution, which is expected to enrich more specific binders, also specifically enriches rapid binders. Supportive SPR experiments have made an important contribution throughout this thesis. The main focus in Chapter 7, however, is on a new application of SPR. The development of a capture approach for supercoiled plasmid DNA, using a triple helix forming oligonucleotide, is described. It could be demonstrated that plasmid DNA can indeed be captured and that SPR can subsequently be used to derive kinetic parameters of a specific interaction with a plasmid. In this particular case the interaction between Lac repressor and its plasmid-based operator was characterised, showing that the association and dissociation rates are ~18 times lower, but that the affinity is the same, when compared to binding to linear operator DNA. This difference underscores the importance of using a DNA substrate with a physiologically relevant topology for studying DNA-protein interactions.</p

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases

Alternative affinity tools: more attractive than antibodies?

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future development

Alternative affinity tools: more attractive than antibodies?

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future development

A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide

Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interaction

Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this stud

Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this stud