Generation of high-affinity DNA aptamers using an expanded genetic alphabet.
TL;DR: Results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.
Abstract: DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.
Citations
More filters
04 Oct 2017
TL;DR: A critical analysis of the first 25 years of aptamer research is provided to provide suggestions for choosing chemical modifications that can lead to enhanced activity or stability, and propose standards for the characterization of aptamers in the scientific literature.
Abstract: Aptamers are nucleic acid molecules that mimic antibodies by folding into complex 3D shapes that bind to specific targets. Although some aptamers exist naturally as the ligand-binding elements of riboswitches, most are generated in vitro and can be tailored for a specific target. Relative to antibodies, aptamers benefit from their ease of generation, low production cost, low batch-to-batch variability, reversible folding properties and low immunogenicity. However, the true value of aptamers lies in the simplicity by which these molecules can be engineered into sensors, actuators and other devices that are often central to emerging technologies. This Review examines changing trends in aptamer technology by analysing the first quarter century of aptamer data that is available in the scientific literature (1990–2015). We highlight specific examples that showcase the use of aptamers in key applications, discuss challenges that have impeded the success of aptamers in practical applications, provide suggestions for choosing chemical modifications that can lead to enhanced activity or stability, and propose standards for the characterization of aptamers in the scientific literature. Aptamers are nucleic acid molecules that can be evolved to bind to specific molecular targets and have found applications in technologies such as sensors and actuators. This Review provides a critical analysis of the first 25 years of aptamer research.
474 citations
TL;DR: The effect of various functional groups at the 5-position of uracil is explored and it is found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers).
Abstract: Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the “diversity gap” between nucleic acid–based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics.
385 citations
TL;DR: This review provides update on the recent advances in this rapidly progressing field of research with particular emphasis on generation of aptamers and their applications in biosensing, biotechnology and medicine.
Abstract: Aptamers are short, single-stranded DNA, RNA, or synthetic XNA molecules that can be developed with high affinity and specificity to interact with any desired targets. They have been widely used in facilitating discoveries in basic research, ensuring food safety and monitoring the environment. Furthermore, aptamers play promising roles as clinical diagnostics and therapeutic agents. This review provides update on the recent advances in this rapidly progressing field of research with particular emphasis on generation of aptamers and their applications in biosensing, biotechnology and medicine. The limitations and future directions of aptamers in target specific delivery and real-time detection are also discussed.
352 citations
Additional excerpts
...This includes incorporation of hydrophobic base 7-(2-thienyl) imidazo (4,5-b) pyridine (Ds) nucleotides into a random natural nucleotides sequence library to obtain aptamers with the increased affinity [64]....
[...]
TL;DR: DNA- and RNA-like systems built from eight nucleotide “letters” that form four orthogonal pairs that meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal are reported.
Abstract: We report DNA- and RNA-like systems built from eight nucleotide “letters” (hence the name “hachimoji”) that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrodinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.
298 citations
TL;DR: In this review, it is shown that in the recent years a significant progress was done in the EC analysis of practically all proteins, based on electroactivity of amino acid (aa) residues in proteins.
Abstract: In this review, we wish to show that in the recent years a
significant progress was done in the EC analysis of practically
all proteins, based on electroactivity of amino acid (aa)
residues in proteins. Also electrochemistry of polysaccharides,
oligosaccharides and glycoproteins greatly advanced in creating
important steps for its larger application in the glycoprotein
research. In recent decades, a great effort was devoted to the
discovery and application of biomarkers for analysis of
different diseases, including cancer. In the following
paragraphs, special attention will be paid (i) to intrinsic
electroactivity of peptides and proteins, including the
sensitivity to changes in protein 3D structures, as well as to
recent advances in EC investigations of DNA-protein
interactions, (ii) to intrinsic electroactivity of glycans and
polysaccharides, advances in EC detection of
lectin-glycoprotein interactions and to introduction of
electroactive labels to polysaccharides and glycans and finally
(iii) to EC detection of protein biomarkers, based
predominantly on application of antibodies in immunoassays,
nucleic acid and peptide aptamers for construction of
aptasensors, and lectin biosensors for detection of
glycoprotein biomarkers.
260 citations
References
More filters
TL;DR: High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species.
Abstract: High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
9,367 citations
TL;DR: Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules.
Abstract: Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
8,781 citations
TL;DR: A series of aptamers currently in development may change how nucleic acid therapeutics are perceived and will increasingly find use in concert with other therapeutic molecules and modalities.
Abstract: Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.
1,707 citations
TL;DR: Pegaptanib was shown in clinical trials to be effective in treating choroidal neovascularization associated with age-related macular degeneration and has the notable distinction of being the first aptamer therapeutic approved for use in humans, paving the way for future aptamer applications.
Abstract: Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Pegaptanib sodium (Macugen; Eyetech Pharmaceuticals/Pfizer) is an RNA aptamer directed against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for pathological ocular neovascularization and vascular permeability. After nearly a decade of preclinical development to optimize and characterize its biological effects, pegaptanib was shown in clinical trials to be effective in treating choroidal neovascularization associated with age-related macular degeneration. Pegaptanib therefore has the notable distinction of being the first aptamer therapeutic approved for use in humans, paving the way for future aptamer applications.
1,327 citations
TL;DR: A versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations, which will enable the discovery of novel biomarkers in a manner that is unencumbered by the incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.
Abstract: Background
The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.
1,245 citations