post-SELEX Modification Research Articles

Post-SELEX modifications with locked nucleic acids (LNA) of a SEA-specific DNA aptamer assisted by in silico modelling

Post-SELEX modifications assisted by in silico modelling are powerful tools to improve the performance of aptamers, by providing a rational approach for the selection of modified-versions.

Interrogating Aptamer Chemical Space Through Modified Nucleotide Substitution Facilitated by Enzymatic DNA Synthesis.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids.

Electrochemical Aptasensors for Antibiotics Detection: Recent Achievements and Applications for Monitoring Food Safety.

Antibiotics are often used in human and veterinary medicine for the treatment of bacterial diseases. However, extensive use of antibiotics in agriculture can result in the contamination of common food staples such as milk. Consumption of contaminated products can cause serious illness and a rise in antibiotic resistance. Conventional methods of antibiotics detection such are microbiological assays chromatographic and mass spectroscopy methods are sensitive; however, they require qualified personnel, expensive instruments, and sample pretreatment. Biosensor technology can overcome these drawbacks. This review is focused on the recent achievements in the electrochemical biosensors based on nucleic acid aptamers for antibiotic detection. A brief explanation of conventional methods of antibiotic detection is also provided. The methods of the aptamer selection are explained, together with the approach used for the improvement of aptamer affinity by post-SELEX modification and computer modeling. The substantial focus of this review is on the explanation of the principles of the electrochemical detection of antibiotics by aptasensors and on recent achievements in the development of electrochemical aptasensors. The current trends and problems in practical applications of aptasensors are also discussed.

Chemical Modifications for a Next Generation of Nucleic Acid Aptamers.

In the past three decades, in vitro systematic evolution of ligands by exponential enrichment (SELEX) has yielded many aptamers for translational applications in both research and clinical settings. Despite their promise as an alternative to antibodies, the low success rate of SELEX (∼30 %) has been a major bottleneck that hampers the further development of aptamers. One hurdle is the lack of chemical diversity in nucleic acids. To address this, the aptamer chemical repertoire has been extended by introducing exotic chemical groups, which provide novel binding functionalities. This review will focus on how modified aptamers can be selected and evolved, with illustration of some successful examples. In particular, unique chemistries are exemplified. Various strategies of incorporating modified building blocks into the standard SELEX protocol are highlighted, with a comparison of the differences between pre-SELEX and post-SELEX modifications. Nucleic acid aptamers with extended functionality evolved from non-natural chemistries will open up new vistas for function and application of nucleic acids.

Modelling aptamers with nucleic acid mimics (NAM): From sequence to three-dimensional docking.

Aptamers are single-stranded oligonucleotides, formerly evolved by Systematic Evolution of Ligands by EXponential enrichment (SELEX), that fold into functional three-dimensional structures. Such conformation is crucial for aptamers’ ability to bind to a target with high affinity and specificity. Unnatural nucleotides have been used to develop nucleic acid mimic (NAM) aptamers with increased performance, such as biological stability. Prior knowledge of aptamer-target interactions is critical for applying post-SELEX modifications with unnatural nucleotides since it can affect aptamers’ structure and performance. Here, we describe an easy-to-apply in silico workflow using free available software / web servers to predict the tertiary conformation of NAM, DNA and RNA aptamers, as well as the docking with the target molecule. Representative 2ʹ-O-methyl (2ʹOMe), locked nucleic acid (LNA), DNA and RNA aptamers, with experimental data deposited in Protein Data Bank, were selected to validate the workflow. All aptamers’ tertiary structure and docking models were successfully predicted with good structural similarity to the experimental data. Thus, this workflow will boost the development of aptamers, particularly NAM aptamers, by assisting in the rational modification of specific nucleotides and avoiding trial-and-error approaches.

Expanding the recognition interface of the thrombin-binding aptamer HD1 through modification of residues T3 and T12

Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands.

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5′-GGT TGG TGT GGT TGG-3′ that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent’s aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Derivatizations of Sgc8-c aptamer to prepare metallic radiopharmaceuticals as imaging diagnostic agents: Syntheses, isolations, and physicochemical characterizations.

Aptamers, oligonucleotides with the capability to bind to a target through non-covalent bonds with high affinity and specificity, have a great number of advantages as scaffold to prepare molecular imaging agents. In this sense, we have performed post-SELEX modifications of a truncated aptamer, Sgc8-c, which bind to protein tyrosine kinase 7 to obtain a specific molecular targeting probe for in vivo diagnosis and in vivo therapy. Herein, we describe the synthetic efforts to prepare conjugates between Sgc8-c and different metallic ions chelator moieties in short times, high purities, and adequate yields. The selected chelator moieties, derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 2-benzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid, and 6-hydrazinonicotinic acid, were covalently attached at the 5'-aptamer position yielding the expected products which were stable in aqueous solution up to 75°C and in typical aptamer storage conditions at least for 30days.

An Update on Aptamer-Based Multiplex System Approaches for the Detection of Common Foodborne Pathogens

Foodborne ailments constitute a public health challenge and pose an incredible economic burden in healthcare system around the globe. This dilemma has urged authorities and other entities working in field of food quality control and supply chain to play a pivotal role in ensuring food safety. Analytical strategies have been developed using numerous systematic evolution of ligands by exponential enrichment (SELEX) methods to assure food safety. High-affinity and high-sensitivity ssDNA and RNA aptamers against pathogens have emerged as a novel strategy, as compared to the more resource-demanding and complicated biochemical test-based approaches. Thus, this review aims to focus on some methods used in the selection of specific bare, modified, and conjugated aptamers and on the further analysis of selected aptamers using flow cytometer or post-SELEX modifications for enhanced detection of frequently diagnosed foodborne bacteria such as Bacillus sp., Campylobacter jejuni, Escherichia sp., Salmonella sp., Staphylococcus aureus, Shigella sp., Listeria monocytogenes, and Streptococcus pyogenes and/or targeting their cell components towards attaining fast, sensitive, and selective methods for the detection of pathogens in food(s) or other sources.

The Toolbox for Modified Aptamers.

Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.

ChemInform Abstract: Dressed for Success — Applying Chemistry to Modulate Aptamer Functionality

AbstractReview: 70 refs.

Dressed for success – applying chemistry to modulate aptamer functionality

Aptamers entered the stage in the early 1990s. Since then they have proven to be versatile tools for molecular biology and biomedical sciences. The combination of chemical synthesis with aptamer generation and post-SELEX modification now provides means for adapting these nucleic acids to almost every desired application and to tune their properties as required. In this review we discuss chemical approaches towards aptamer variation.

Locked Nucleic Acid and Aptamers

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of adopting well-defined 3-dimensional shapes, which enables targeting of peptides, proteins, small molecules, and live cells (MAYER, 2009; THIEL, 2009, Keefe et al., 2010). Aptamers are typically generated by in vitro selection using the process known as SELEX (systematic evolution of ligands by exponential enrichment) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). SELEX involves iterative rounds of selection and polymerase-catalyzed enrichment using polymerase chain reaction (PCR) of bound aptamers from a large library of typically ∼80-nucleotide-long nucleic acid sequences composed of a central randomized region flanked by two primer-binding regions. Aptamers with nanomolar binding constants against their targets have frequently been evolved, and aptamers can therefore be considered “the antibodies of the nucleic acid world.” Even though the use of aptamers as therapeutic agents is promising, there are challenges that need to be met. One is that the size and polyanionic character of aptamers limits intracellular delivery. However, recent in vivo results on aptamer-mediated specific delivery of an anti-cancer small interfering RNA (siRNA) to prostate cancer cells (Dassie et al., 2009) have not only underlined the potential applicability of aptamers as drug delivery constructs but also give hope for intracellular applications of therapeutic aptamers. Importantly, aptamers, contrary to siRNA and antisense constructs, obviously can be applied against extracellular targets. Size is not only an obstacle for intracellular delivery, but also poses a challenge in manufacturing, as current production technology relies on DNA synthesizers for automated solid support-based synthesis. Another challenge is biodistribution, as unconjugated oligonucleotides are rapidly excreted via the kidneys upon intravenous administration. Conjugation with polyethyene glycol (PEG) units (Veronese and Pasut, 2005) is an approach that has been applied to improve biodistribution of aptamers. The SELEX procedure also can be limiting, as it is time consuming if performed in the standard manual fashion. Automation may in some cases alleviate this challenge (Eulberg et al., 2005; Wochner et al., 2007), and alternative selection/evolution strategies are being developed (Drabovich et al., 2006; Nitsche et al., 2007; Aquino-Jarquin and Toscano-Garibay, 2011; Arnold et al., 2012). One aptamer has been approved as drug (Pegaptanib) to treat age-related macular degeneration, and others are, or have been, in various stages of clinical development (FAMULOK, 2009; Keefe et al., 2010). In general, the therapeutic candidates have been obtained by post-SELEX modification of aptamers evolved by a full SELEX procedure including 10–20 rounds of selection and enrichment. Post-SELEX modification typically involves truncation into shorter aptamer candidates, conjugation (e.g., PEGylation) for improved biodistribution, and incorporation of chemically modified nucleotides for improved biostability. Post-SELEX chemical modification is necessary, as rather few modified nucleoside triphosphates, for example, 2′-fluoro-RNA, 2′-amino-RNA and 5-substituted pyrimidine nucleoside triphosphates (MAYER, 2009; Lauridsen et al., 2012), are substrates for the polymerase-catalyzed reactions required for efficient SELEX procedures. Post-SELEX modifications are performed in iterative rounds of synthesis and biological evaluation to ensure that modifications are compatible with the desired aptamer properties. A prominent nucleotide modification in relation to nucleic acid drug discovery is locked nucleic acid (LNA, Fig. 1) (Imanishi and Obika, 2002; Jepsen and Wengel, 2004; Veedu and Wengel, 2010). Incorporation of LNA nucleotides into DNA or RNA strands induces unrivalled increases in duplex thermal stabilities, and LNA phosphorothioate oligonucleotides have shown unique characteristics as single-stranded antisense molecule targeting RNA, for example, messenger RNA or microRNA (Jepsen and Wengel, 2004; Lanford et al., 2010). LNA nucleotides increase nucleolytic stability of oligonucleotides, and their pronounced duplex-stabilizing effect furthermore renders LNA-modified siRNA duplexes highly biostable (Glud et al., 2009). FIG. 1. Chemical structure of DNA, RNA, and locked nucleic acids (LNA). In our research group, we are currently working on development and evolution of LNA-containing aptamers (Veedu and Wengel, 2009). We want to explore LNA toward a breakthrough within the aptamer field. As mentioned above, despite obvious benefits and two decades of research and development, only relatively few aptamers are currently undergoing clinical development. We therefore think that the need for new approaches and methods to obtain short aptamers with increased biostability, structural stability, and biological function is evident. Post-SELEX LNA modification of aptamers as summarized below have been reported to yield some of these desired properties, but we simultaneously focus on enabling efficient and reliable de novo evolution of LNA aptamers.

SPR evaluation of binding kinetics and affinity study of modified RNA aptamers towards small molecules

Establishing an efficient method for evaluating the affinity changes after post-SELEX modification of aptamers is essential for broadening the application of these oligonucleotides in biosensing. This is especially challenging when the ligand is a small molecule. Changes in affinity upon partial or total replacement of 2′–OH with 2′-OMe groups in the ribose moieties of a tobramycin binding RNA aptamer are described. The kinetic profile and binding properties of the different anti-tobramycin aptamers were measured by surface plasmon resonance (SPR) experiments through a real-time binding assay with the antibiotic covalently coupled to the gold sensor. This configuration maximizes the changes associated to the recognition event, which is otherwise undetectable. The results indicated that the modification slightly affects the binding characteristics of the parent RNA, while conferring biological stability to the aptamers against nucleases.

A Mini-Library of TBA Analogues Containing 3′-3′ and 5′-5′ Inversion of Polarity Sites

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3′-3′ or a 5′-5′ inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.

Post-SELEX combinatorial optimization of aptamers

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor

Background: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a potent inducer of new blood vessel growth (angiogenesis) that contributes to the pathology of many angiogenesis-associated disease states such as psoriasis, rheumatoid arthritis and cancer. Few molecular entities capable of binding to VPF/VEGF with high affinity and specificity have been described to date. Results: Nuclease-resistant 2′-amino-2′-deoxypyrimidine nucleotide RNA (2′-aminopyrimidine RNA) ligands that bind to VPF/VEGF with high affinity have been identified by iterative rounds of affinity-selection/amplification from two independent random libraries. The sequence information that confers high affinity binding toVPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5′- CCCUGAUGGUAGACGCCGGGGUG-3′ (2′-aminopyrimidine nucleotides are designated with italic letters). Of the 14 ribopurines in this minimal ligand, 10 can be substituted with the corresponding 2′-O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGF. In fact, the 2′ O-methyl substitution at permissive positions leads to a ∼17-fold improvement in the binding affinity to VPF/VEGF The higher affinity results from the reduction in the dissociation rate constant of the 2′-O-methyl-substituted RNA ligand from the protein compared to the unsubstituted ligand. The 2′-O-methyl-substituted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resistance of the ligand is further improved by the 2′-O-methyl substitutions and the addition of short phosphorothioate caps to the 3′- and 5′-ends. Conclusions: We have used the SELEX ( s ystematic e volution of ligands by e xponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.