Aptamers as therapeutic and diagnostic agents

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX. Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2'OH groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. A new approach to diagnostics is also described. Aptamer arrays on solid surfaces will become available rapidly because the SELEX protocol has been successfully automated. The use of photo-cross-linkable aptamers will allow the covalent attachment of aptamers to their cognate proteins, with very low backgrounds from other proteins in body fluids. Finally, protein staining with any reagent which distinguishes functional groups of amino acids from those of nucleic acids (and the solid support) will give a direct readout of proteins on the solid support.     

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-kappaB

A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-kappaB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-kappaB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-kappaB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer's relative affinity to basic groups in proteins such as NF-kappaB by helping to "strip" the cations from the aptamer backbone.     

Beyond antibodies: New affinity reagents to unlock the proteome

Antibodies have been the workhorse reagents of protein capture and quantification since their 1959 debut in the RIAs developed by Yalow and Berson. However, there are technical challenges to the use of antibodies in highly multiplexed arrays aimed at measuring hundreds or even thousands of proteins at one time. We describe here a recently developed class of synthetic protein‐binding reagents (slow off‐rate modified aptamer). We discuss the chemical makeup and protein binding specifications of slow off‐rate modified aptamer reagents, compare them to traditional aptamers and antibodies, briefly describe the novel proteomic assay that takes advantage of their unique properties, and provide several examples of their multiple applications to biomarker discovery and validation across a range of biomedical science questions.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.     

Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics

EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organization. It binds directly to microtubule plus ends and recruits other plus end–localizing proteins. Most EB1-binding proteins contain a Ser–any residue–Ile-Pro (SxIP) motif. Here we describe the isolation of peptide aptamers with optimized versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognized as a powerful technique. We show that SxIP aptamers can bind microtubule plus ends in cells and functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. We also identify aptamers binding to human EB1 and EB3, which have sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.     

Comprehensive proteomic analysis of mineral nanoparticles derived from human body fluids and analyzed by liquid chromatography-tandem mass spectrometry

Mineralo-protein nanoparticles (NPs) formed spontaneously in the body have been associated with ectopic calcifications seen in atherosclerosis, chronic degenerative diseases, and kidney stone formation. Synthetic NPs are also known to become coated with proteins when they come in contact with body fluids. Identifying the proteins found in NPs should help unravel how NPs are formed in the body and how NPs in general, be they synthetic or naturally formed, interact within the body. Here, we developed a proteomic approach based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to determine the protein composition of carbonate-apatite NPs derived from human body fluids (serum, urine, cerebrospinal fluid, ascites, pleural effusion, and synovial fluid). LC–MS/MS provided not only an efficient and comprehensive determination of the protein constituents, but also a semiquantitative ranking of the identified proteins. Notably, the identified NP proteins mirrored the protein composition of the contacting body fluids, with albumin, fetuin-A, complement C3, α-1-antitrypsin, prothrombin, and apolipoproteins A1 and B-100 being consistently associated with the particles. Since several coagulation factors, calcification inhibitors, complement proteins, immune regulators, protease inhibitors, and lipid/molecule carriers can all become NP constituents, our results suggest that mineralo-protein complexes may interface with distinct biochemical pathways in the body depending on their protein composition. We propose that LC–MS/MS be used to characterize proteins found in both synthetic and natural NPs.     

Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization

We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.     

Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins

Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody) can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and quality control setting. The technique can provide an early warning of structural changes during the manufacturing process that could have consequential outcomes downstream.     

Crystal Structure of Interleukin-6 in Complex with a Modified Nucleic Acid Ligand

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (K_d = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (K_d = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (K_d = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.     

High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands

We have isolated 2′-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of SA-binding aptamers. In order to provide a general method for the exploitation of these aptamers, we produced derivatives in which they were fused to the naturally structured RNA elements, CopT or CopA. In parallel, we produced derivatives of CD4-binding aptamers fused to the complementary CopA or CopT elements. When mixed, these two chimeric aptamers rapidly hybridized, by virtue of CopA–CopT complementarity, to form stable, bi-functional aptamers that we called ‘adaptamers’. We show that a CD4–SA-binding adaptamer can be used to capture CD4 onto a SA-derivatized surface, illustrating their general utility as indirect affinity ligands.     

Using indirect protein-protein interactions for protein complex prediction

Protein complexes are fundamental for understanding principles of cellular organizations. As the sizes of protein-protein interaction (PPI) networks are increasing, accurate and fast protein complex prediction from these PPI networks can serve as a guide for biological experiments to discover novel protein complexes. However, it is not easy to predict protein complexes from PPI networks, especially in situations where the PPI network is noisy and still incomplete. Here, we study the use of indirect interactions between level-2 neighbors (level-2 interactions) for protein complex prediction. We know from previous work that proteins which do not interact but share interaction partners (level-2 neighbors) often share biological functions. We have proposed a method in which all direct and indirect interactions are first weighted using topological weight (FS-Weight), which estimates the strength of functional association. Interactions with low weight are removed from the network, while level-2 interactions with high weight are introduced into the interaction network. Existing clustering algorithms can then be applied to this modified network. We have also proposed a novel algorithm that searches for cliques in the modified network, and merge cliques to form clusters using a "partial clique merging" method. Experiments show that (1) the use of indirect interactions and topological weight to augment protein-protein interactions can be used to improve the precision of clusters predicted by various existing clustering algorithms; and (2) our complex-finding algorithm performs very well on interaction networks modified in this way. Since no other information except the original PPI network is used, our approach would be very useful for protein complex prediction, especially for prediction of novel protein complexes.     

Flavin recognition by an RNA aptamer targeted toward FAD

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

Background

DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification.

Methodology

We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit⁺ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types.

Conclusions/Significance

Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).     

RNA aptamers to S-adenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody

To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.