Aptamers are single-stranded oligonucleotides that fold into defined architectures and bind to targets such as proteins. In binding proteins they often inhibit protein–protein interactions and thereby may elicit therapeutic effects such as antagonism. Aptamers are discovered using SELEX (systematic evolution of ligands by exponential enrichment), a directed in vitro evolution technique in which large libraries of degenerate oligonucleotides are iteratively and alternately partitioned for target binding. They are then amplified enzymatically until functional sequences are identified by the sequencing of cloned individuals. For most therapeutic purposes, aptamers are truncated to reduce synthesis costs, modified at the sugars and capped at their termini to increase nuclease resistance, and conjugated to polyethylene glycol or another entity to reduce renal filtration rates. The first aptamer approved for a therapeutic application was pegaptanib sodium (Macugen; Pfizer/Eyetech), which was approved in 2004 by the US Food and Drug Administration for macular degeneration. Eight other aptamers are currently undergoing clinical evaluation for various haematology, oncology, ocular and inflammatory indications. Aptamers are ultimately chemically synthesized in a readily scalable process in which specific conjugation points are introduced with defined stereochemistry. Unlike some protein therapeutics, aptamers do not elicit antibodies, and because aptamers generally contain sugars modified at their 2′-positions,…
What is an Aptamer? An aptamer is a short, single-stranded oligonucleotide (DNA or RNA) or peptide that binds to a specific target molecule (e.g., proteins, small molecules, cells, viruses) with high affinity and specificity. Often called "chemical antibodies," they offer advantages like stability, low-cost synthesis, and minimal batch-to-batch variation. The Core Process: SELEX The standard method for aptamer selection is SELEX (Systematic Evolution of Ligands by EXponential enrichment). Basic SELEX Workflow: Library Synthesis: Create a vast random-sequence oligonucleotide library (typically 10¹³ - 10¹⁵ unique sequences) flanked by constant primer regions for PCR amplification. Incubation: The library is incubated with the target molecule under controlled conditions (buffer, temperature, time). Partitioning: Bound sequences are separated from unbound ones. This is the most critical step and varies based on target (e.g., filtration, affinity columns, magnetic bead separation). Elution: Bound aptamers are recovered from the target (e.g., by denaturation or competitive elution). Amplification: The recovered pool is amplified by PCR (for DNA) or RT-PCR (for RNA) to create an enriched library for the next round. Iteration: Steps 2-5 are repeated (typically 8-15 rounds) to progressively enrich for sequences with the highest affinity and specificity. Cloning & Sequencing: The final enriched pool is cloned and sequenced to identify individual aptamer candidates. Key Variants of…
The unique secondary and tertiary structures of aptamers provide the specificity to detect even small structural changes in the target molecule, including the presence or absence of methyl or hydroxyl groups or differences in enantiomeric configurations. Aptamers that bind specific targets are identified through a process known as Systematic Evolution of Ligands by Exponential enrichment (SELEX) in which binding molecules are selected from a large and diverse library of nucleic acids (either DNAs or RNAs). In this process, the nucleic acid library is incubated with the target molecule. Non-binding nucleic acids are then washed away, leaving behind only the molecules that have a capacity to bind to the target molecule. The nucleic acids that are not washed away are then used to create a new library of nucleic acids that is enriched for the subset that binds the desired target. Repeating this selection-cycle on each subsequent library with increasing stringency of binding (e.g. lower concentration of target), ensures that nucleic acids that bind to the target with both high specificity and high affinity are enriched. Aptamers are short, single-stranded oligonucleotides (DNA or RNA) that bind to specific target molecules with high affinity and specificity. They are often called "chemical antibodies."…
Negative aptamer selection—often called negative selection or counter-selection—is a deliberate filtering step in SELEX(Systematic Evolution of Ligands by EXponential enrichment) designed to remove sequences that bind to the wrong things. Instead of enriching binders to your intended target, negative selection enriches your final pool for what you actually want in real-world use: high specificity, low background, and minimal cross-reactivity. In modern aptamer discovery, negative selection is not “optional polish.” It is one of the most effective ways to prevent selection artifacts—like aptamers that bind to beads, linkers, tags, surfaces, common matrix components, or closely related off-target molecules—from dominating your pool. 1) What “Negative Aptamer Selection” Means (and Why It Exists) During SELEX, you start with a huge randomized DNA/RNA library and iteratively enrich sequences that bind. The catch is that many sequences bind strongly to unintended components in the experimental system: immobilization substrates (e.g., beads, membranes) affinity tags or capture molecules (e.g., streptavidin–biotin systems) blockers, serum proteins, plastic, or assay buffers structurally similar molecules (analogs) that you must not bind Negative selection introduces a decoy binding step: you expose the library to an unwanted target (or “negative target”), then discard the sequences that bind it and keep…
“Completion of SELEX” refers to the point in the Systematic Evolution of Ligands by EXponential enrichment (SELEX)workflow where iterative selection rounds have produced an enriched nucleic-acid pool (DNA or RNA) that contains high-affinity, high-specificity binding sequences (aptamers) for a defined target, and further rounds provide diminishing improvements. In practical terms, completion is less a single universal round number and more a decision point supported by enrichment evidence, performance metrics, and downstream readiness. 1) SELEX in One Picture (Why “Completion” Exists at All) SELEX is an iterative evolutionary loop performed in vitro: Start with a diverse library (randomized nucleic-acid sequences). Bind the library to a target (protein, small molecule, cell surface, complex mixture, etc.). Partition: separate binders from non-binders (the critical “selection” step). Elute and amplify the binders (PCR for DNA; RT-PCR for RNA). Repeat with increasing stringency (less target, harsher washes, counter-selection, etc.). “Completion” matters because every additional round costs time, introduces amplification bias, and can over-enrich “fast amplifiers” rather than “best binders.” Modern practice treats completion as an optimization endpoint, not a ritual number of rounds. 2) What “Completion of SELEX” Typically Means (Conceptual Definition) A common knowledge-centered definition is: The pool has converged toward one…
What “SELEX Aptamer Selection” Means SELEX stands for Systematic Evolution of Ligands by Exponential Enrichment. In plain terms, SELEX aptamer selectionis an iterative laboratory workflow that starts with a huge pool of random DNA or RNA sequences and repeatedly enriches the fraction that binds a chosen target with high affinity and specificity. The “winners” are called aptamers—single-stranded nucleic acids that fold into 3D shapes capable of target recognition, often compared to “chemical antibodies,” but made by selection and synthesis rather than immune systems. Core Concept: Darwinian Evolution in a Test Tube SELEX is essentially variation + selection + amplification: Variation: Begin with a randomized oligonucleotide library (often ~10^13–10^16 unique sequences). Selection: Expose the library to the target; keep sequences that bind. Amplification: PCR (or RT-PCR for RNA workflows) amplifies binders to create the next-round pool. Increasing stringency: Each round tightens conditions (less target, harsher washes, more competitors), enriching the best binders over multiple cycles. Most conventional SELEX workflows run multiple rounds (often roughly 6–15) before candidates are sequenced and characterized. The Classic SELEX Workflow (Step-by-Step, With the “Why”) 1) Library design (the “starting universe”) A typical library contains: A random region (e.g., N30–N60) that can…
