Aptamer affinity optimization refers to the process of improving the binding strength and specificity of an aptamer—a short, single-stranded DNA or RNA molecule—to its target molecule (protein, small molecule, or cell surface marker). Higher affinity aptamers result in better sensitivity and selectivity in diagnostic, therapeutic, and research applications. Key Concepts Affinity vs. Specificity Affinity: How tightly an aptamer binds to its target (quantified by dissociation constant, K_d). Lower K_d indicates higher affinity. Specificity: Aptamer’s ability to distinguish the target from similar molecules. Factors Affecting Aptamer Affinity Sequence composition and length. Secondary and tertiary structures (e.g., stem-loops, G-quadruplexes). Target-binding site accessibility. Ionic conditions (Mg²⁺, Na⁺) and pH. Optimization Strategies In vitro Evolution Methods SELEX (Systematic Evolution of Ligands by EXponential enrichment) Iterative rounds of selection and amplification to enrich high-affinity sequences. Variants: High-stringency SELEX: Lower target concentrations or harsher washing steps. Counter-SELEX: Remove sequences binding to similar molecules to enhance specificity. Truncation and Structural Optimization Remove non-essential nucleotides to reduce size while retaining binding. Stabilize key secondary structures (e.g., adding stem loops or G-quadruplex motifs). Chemical Modifications 2’-Fluoro, 2’-O-methyl nucleotides: Enhance stability and sometimes affinity. PEGylation or LNA (locked nucleic acids): Improve folding and binding. Rational Design & Mutagenesis Identify and…
An aptamer library is a diverse pool of nucleic acid sequences (DNA or RNA) from which specific aptamers—short oligonucleotides that bind to target molecules with high affinity—can be selected. Constructing a high-quality library is the foundation of aptamer screening technologies like SELEX. 2. Key Components of an Aptamer Library Randomized Region The central portion of the aptamer, typically 20–60 nucleotides, is randomized to generate diversity. Example: N20–N40 where N = A, T/U, G, or C. The diversity determines the probability of finding high-affinity binders. Flanking Constant Regions Short sequences (~15–25 nt) at both ends of the randomized region. Functions: Primer binding sites for PCR amplification. Stability and structural constraints. Overall Length Usually 40–100 nucleotides, balancing structural complexity and amplification efficiency. 3. Steps of Library Construction Design of Oligonucleotides Include random regions flanked by known primer sequences. Example structure:5'-[Primer]-N40-[Primer]-3' Chemical Synthesis Use solid-phase DNA/RNA synthesis to generate the oligonucleotides. Random nucleotides are incorporated using a controlled mixture of A, T/U, G, C. Amplification (for DNA libraries) PCR amplifies the synthesized sequences. RNA libraries require in vitro transcription from DNA templates. Purification Remove truncated or incomplete sequences. Methods: PAGE purification or HPLC. Quality Control Ensure correct length, diversity, and absence of biases.…
“High-throughput aptamer screening” is a method used to rapidly identify aptamers—short single-stranded DNA or RNA molecules—that can bind specifically to a target molecule, such as a protein, small molecule, or even whole cells. Let’s break this down in detail: 1. What Are Aptamers? Aptamers are oligonucleotides (DNA or RNA) that fold into specific three-dimensional shapes allowing them to bind with high affinity and specificity to their targets. They function similarly to antibodies but are synthetic, smaller, more stable, and can be chemically modified. 2. High-Throughput Screening (HTS) in Aptamer Discovery Traditional aptamer discovery uses SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which involves multiple iterative rounds of binding, separation, and amplification. High-throughput aptamer screening accelerates this process by using automation and large-scale technologies to simultaneously test thousands to millions of sequences against the target. 3. Key Techniques in High-Throughput Aptamer Screening Microarray-Based Screening Thousands of aptamer candidates are immobilized on a chip. The target (protein, small molecule, or cell) is fluorescently labeled and applied. Aptamers that bind the target emit signals detected by imaging. Next-Generation Sequencing (NGS)-Coupled SELEX After each SELEX round, sequences are analyzed via NGS. Sequence enrichment patterns reveal high-affinity aptamer candidates without the need for extensive…
1. What is SELEX? SELEX stands for Systematic Evolution of Ligands by EXponential enrichment. It is a laboratory technique used to identify aptamers—short single-stranded DNA or RNA sequences that can bind specifically to a target molecule (proteins, small molecules, cells, or even viruses). Aptamers act similarly to antibodies but are synthetic, highly stable, and can be chemically modified. 2. Principle of SELEX The SELEX process is based on iterative rounds of selection and amplification: Library Preparation Start with a large randomized pool of oligonucleotides (typically 10^13–10^15 unique sequences). Each sequence is a potential aptamer candidate. Binding (Target Incubation) Incubate the library with the target molecule. Only sequences that can bind the target will stay attached; non-binders are washed away. Partitioning (Separation of Binders and Non-binders) Physically separate bound sequences from unbound sequences. Techniques depend on the target (magnetic beads, affinity columns, etc.). Elution Bound sequences are eluted (released) from the target. Amplification The eluted sequences are amplified using PCR (for DNA aptamers) or RT-PCR (for RNA aptamers). This generates an enriched pool for the next round. Iterative Rounds Steps 2–5 are repeated for 8–15 rounds to gradually enrich sequences with high affinity and specificity for the target. Sequence Identification After…
Core Principle & Typical Service Workflow A professional service for Conventional SELEX typically follows this established, iterative cycle (8-15 rounds), as visualized below: Key Service Characteristics Target Immobilization: The target molecule is fixed to a solid support (e.g., magnetic beads, column resin, nitrocellulose membrane). Positive Selection: The library is passed over the immobilized target. Sequences with some binding affinity are retained, while others are washed away. Stringency Control: The service provider systematically increases selection pressure across rounds (e.g., by reducing target concentration, increasing wash stringency, adding counter-targets in Negative SELEX steps) to drive the evolution of high-affinity, specific binders. Monitoring: Enrichment is tracked via quantitative PCR, and final pools are analyzed by Next-Generation Sequencing (NGS) to identify convergent sequence families. Common Applications for this Service This classic approach is ideal for: Proteins that are stable and can be immobilized without losing native conformation (e.g., antibodies, enzymes, recombinant tags). Large molecules or complexes (e.g., viruses, whole cells—though Cell-SELEX is now more common for cells). Establishing proof-of-concept for a new target class where simpler, robust methodology is preferred. Deliverables Similar to other SELEX services, clients receive: A final report detailing the selection process and conditions. A list of top-ranked aptamer sequences with affinity (Kd) and specificity data. Aliquots of synthesized, validated…
What is Filter Membrane Binding SELEX? SELEX (Systematic Evolution of Ligands by EXponential enrichment) is the standard method for discovering high-affinity, specific nucleic acid aptamers. The Filter Membrane Binding variant is one of the most classic and robust SELEX techniques. Core Principle: It leverages a nitrocellulose or mixed cellulose ester filter membrane, which irreversibly binds proteins and other macromolecules but allows short, unbound single-stranded DNA or RNA oligonucleotides to pass through. The Selection Mechanism: During each selection round, the target molecule (e.g., a protein) is immobilized on the filter. An immense library of random oligonucleotides (10^13 - 10^15 unique sequences) is applied. Only sequences that bind to the target are retained on the filter with it. Unbound sequences are washed away. The bound aptamer candidates are then eluted, amplified by PCR (or RT-PCR for RNA), and used as the enriched library for the next round. Key Features of the Service A professional service will typically offer: Target Flexibility: Optimal for purified proteins (recombinant or native), protein complexes, viruses, and even some small molecules if conjugated to a carrier protein. Counter-SELEX: A critical step to ensure specificity. The enriched library is passed through a filter bound to non-target molecules (e.g., related proteins, cell lysates, immobilization matrix) to subtract cross-reactive binders. High-Throughput…
What is a Stem Cell Aptamer Screening Service? It is a contract research service where a specialized lab uses Systematic Evolution of Ligands by EXponential Enrichment (SELEX) to discover and develop DNA or RNA aptamers that bind with high affinity and specificity to a target of your choice related to stem cells. Aptamers are often called "chemical antibodies." They are short, single-stranded oligonucleotides that fold into unique 3D shapes, allowing them to bind to targets like proteins, small molecules, or even whole cells. Core Targets for Stem Cell Applications The service can be tailored to screen for aptamers against: Specific Cell Surface Markers: (e.g., CD34, CD133, SSEA-4, TRA-1-60) for identification and isolation. Whole Live Stem Cells: To get aptamers that recognize the unique molecular signature of a specific stem cell type (e.g., mesenchymal stem cells, cancer stem cells, pluripotent stem cells). Differentiation State-Specific Targets: To distinguish between pluripotent, progenitor, and fully differentiated cells. Specific Stem Cell-Derived Products: (e.g., exosomes, vesicles). Typical Workflow of the Service A professional service provider will guide you through these stages: Phase Description Your Input 1. Project Design Defining the target (specific protein, cell line, primary cells), counter-selection cells (to ensure specificity), and desired aptamer properties (e.g., Kd, nuclease resistance). Provide target cells, control cells, and…
Core Concept: Aptamers vs. Antibodies Aptamers are often called "chemical antibodies." Their key advantages for cancer targeting include: Small size: Better tissue penetration. In vitro synthesis: Highly reproducible, no batch-to-batch variation. Ease of modification: Can be chemically tagged with dyes, drugs, or nanoparticles. Low immunogenicity. Target Range: Can bind to proteins, carbohydrates, lipids, or even complex molecular patterns on a whole cell's surface. The Screening Service Workflow (Cell-SELEX) A typical service follows these steps: 1. Project Design & Target Selection Client Input: You define the target (e.g., "Aptamers for metastatic triple-negative breast cancer cell line MDA-MB-231"). Counter-Selection: Crucial step. To ensure specificity, the service provider will also use a control cell line (e.g., normal breast epithelial cells or a less aggressive cancer type) to remove aptamers that bind to common, non-target molecules. Library Design: The provider uses a vast random oligonucleotide library (e.g., 10^14 different sequences). 2. The SELEX Process This is an iterative, multi-round biochemical "fishing" experiment: Incubation: The library is exposed to the target cancer cells. Washing: Weakly or unbound sequences are washed away. Elution: Bound aptamers are recovered (e.g., by heating or trypsinizing cells). Amplification: Recovered aptamers are amplified by PCR (for DNA) or RT-PCR (for RNA). Stringency Increase: In each subsequent round, conditions become stricter (more washing, shorter incubation, addition…
What is the Service? It's the process of using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to identify single-stranded DNA or RNA aptamers that can bind to a target cytokine. The service takes you from target selection to delivering validated aptamer candidates. Standard Workflow (What the Provider Does) Project Scoping & Target Preparation: Target: You specify the cytokine (e.g., TNF-α, IL-6, IFN-γ). The provider may require you to supply the purified, recombinant protein or offer to procure/produce it. Counter-SELEX: A critical step to ensure specificity. The provider will use related proteins (e.g., other cytokines, serum proteins) to eliminate aptamers that bind non-specifically. Library Design & SELEX Cycle: Starts with a vast random oligonucleotide library (10^14 - 10^15 unique sequences). Iterative rounds (8-15+) of: Binding: Incubating the library with the target cytokine. Partitioning: Separating bound from unbound sequences (e.g., via immobilization on beads, filters, or capillary electrophoresis). Amplification: PCR (for DNA) or RT-PCR (for RNA) to enrich the binding sequences. Stringency Increase: Gradually increasing washing rigor and introducing counter-selection to drive selection of high-affinity, specific binders. Next-Generation Sequencing (NGS) & Bioinformatics: After the final rounds, the enriched pool is sequenced using NGS. Bioinformatics tools analyze the data to identify enriched sequence families, consensus motifs, and predict secondary structures.…
What is an Aptamer? An aptamer is a short, single-stranded oligonucleotide (DNA or RNA) that folds into a unique 3D structure, allowing it to bind to a specific target molecule (like a protein) with similar specificity to an antibody. They are often called "chemical antibodies." Why Use a Screening Service Instead of In-House Development? Expertise & Equipment: The screening process (SELEX) requires specialized skills, robotics, and next-generation sequencing (NGS) infrastructure. Time & Cost Efficiency: Outsourcing can be faster and more cost-effective than setting up a new, complex pipeline. Higher Success Rate: Experienced providers have optimized protocols for difficult targets (e.g., membrane proteins, toxic proteins). The Core Process: SELEX The standard method is SELEX (Systematic Evolution of Ligands by EXponential Enrichment). A professional service will offer advanced variants of this process. A Typical Service Workflow: Project Consultation & Design: Target Characterization: Discussion about your protein (purified? membrane-bound? post-translational modifications?). Selection Strategy: Choosing the best SELEX method (e.g., Capillary Electrophoresis-SELEX (CE-SELEX) for very high affinity, Cell-SELEX for cell-surface targets, Toggle-SELEX for cross-species specificity). Counter-Selection: Designing the process to avoid binding to non-target proteins (e.g., carrier proteins, related isoforms). Library Synthesis & Preparation: Creation of a vast random oligonucleotide library (typically 10¹³ - 10¹⁵ unique sequences). The Selection Rounds (Cycles of SELEX): Binding: Incubating the library with the…
