What is a Metal Ion-Targeted Aptamer Screening Service? It is a contract research service where a specialized laboratory uses an in vitro selection process (most commonly SELEX - Systematic Evolution of Ligands by EXponential Enrichment) to identify single-stranded DNA or RNA oligonucleotides (aptamers) that bind with high affinity and specificity to a specific metal ion (e.g., Pb²⁺, Hg²⁺, UO₂²⁺, As³⁺, Cd²⁺). Unlike aptamers for proteins, metal ion aptamers often rely on the ion's unique coordination chemistry to induce a specific fold or structural switch in the oligonucleotide. Core Service Workflow (The Screening Process) A typical service provider would follow these steps: Design & Library Synthesis: Creation of a vast random-sequence oligonucleotide library (10¹⁴ - 10¹⁵ different sequences). Target Preparation: The target (e.g., Pb²⁺) is often presented in a specific buffer system that controls charge, pH, and the presence of competing ions to drive selection for the desired specificity. Selection Rounds (SELEX Cycle): Binding: Incubate the library with the target metal ion. Partition: Separate metal-bound sequences from unbound ones. This is the most critical and challenging step for small ions. Techniques include: Immobilization: Cheating the ion to a solid support (beads). Capture-SELEX: Using a complementary strand or an auxiliary molecule. Size-based separation: If binding induces a conformational change (e.g., dimerization). Amplification: PCR (for…
1. What Are Aptamers? Aptamers are short, single-stranded DNA or RNA oligonucleotides (typically 20-80 bases) that fold into specific 3D structures, allowing them to bind to target molecules (like hormones) with high affinity and specificity, similar to antibodies. They are often called "chemical antibodies." 2. Why Target Hormones with Aptamers? Hormones are critical signaling molecules (e.g., insulin, cortisol, thyroid hormones, estradiol, adrenaline). Aptamers against them offer unique advantages: High Specificity: Can distinguish between structurally similar hormones (e.g., T3 vs. T4). Synthetic & Reproducible: Produced chemically with minimal batch-to-batch variation. Stability: More thermally stable than antibodies. Modifiability: Can be easily labeled with fluorescent dyes, quenchers, or nanoparticles for detection. Low Immunogenicity: Ideal for in vivo diagnostic or therapeutic applications. 3. Core Components of the Screening Service A full-service provider would typically offer the following pipeline: a. Design & Library Construction: Use of a vast random oligonucleotide library (10^14 - 10^15 unique sequences). Customization of library design based on hormone properties (small molecule vs. peptide/protein). b. SELEX Process (The Core Screening): This is an iterative, in vitro selection process. Incubation: The library is exposed to the target hormone (immobilized or in solution). Partitioning: Unbound sequences are washed away; bound sequences (aptamer candidates) are retained. Elution & Amplification: Bound sequences are eluted and amplified by PCR…
What is an Aptamer? Aptamers are single-stranded DNA or RNA oligonucleotides that fold into specific 3D shapes, enabling them to bind to target molecules (proteins, small molecules, cells, viruses) with high affinity and specificity, similar to antibodies. They are often called "chemical antibodies." Why Use Aptamer Screening Services in Drug Discovery? Efficiency: Outsourcing to experts with specialized platforms (SELEX) accelerates discovery. Cost-Effectiveness: Avoids capital investment in complex SELEX and NGS infrastructure. Expertise: Leverages specialized knowledge in oligonucleotide chemistry, bioinformatics, and target biology. Focus: Allows internal teams to concentrate on downstream therapeutic development. Core Components of an Aptamer Screening Service A full-service provider typically offers an end-to-end pipeline: 1. Project Design & Target Preparation Consultation: Defining the target (recombinant protein, cell surface marker, whole cell), desired affinity (nM-pM), and specificity (e.g., against homologs). Counter-SELEX Strategy: Planning to eliminate binders to non-desired epitopes or related targets to ensure high specificity. 2. In Vitro Selection (SELEX) The core technology is SELEX (Systematic Evolution of Ligands by EXponential enrichment). Advanced variants are used for complex targets: Protein-SELEX: For purified recombinant proteins. Cell-SELEX: For membrane proteins in their native conformation on live cells; identifies aptamers for diseased vs. healthy cells. Tissue-SELEX: For even more complex biological environments. Capture-SELEX: For small molecules that are difficult to immobilize. High-Throughput SELEX (HT-SELEX): Uses NGS early…
Core Technology: SELEX The foundation of all these services is the SELEX process, an in vitro method to select aptamers from a vast random library (typically 10^13 - 10^15 unique sequences). The library is incubated with the target, unbound sequences are washed away, and bound sequences are eluted and amplified by PCR (for DNA) or RT-PCR (for RNA). This cycle is repeated 8-15 times to enrich for the tightest binders. Services for Protein Targets This is the most common application, as aptamers are often touted as "chemical antibodies." 1. Standard Protein SELEX: Target: Purified, recombinant proteins (e.g., cytokines, receptors, enzymes, viral capsids). Key Considerations: Protein Purity & Conformation: Critical for success. Services often require >90% purity and verification of native folding. Immobilization: The protein is usually immobilized on beads (e.g., streptavidin/biotin, Ni-NTA/His-tag) to facilitate partitioning. Some services offer solution-phase SELEX to avoid conformation changes. Counter-Selection: To ensure specificity, libraries are pre-incubated with related proteins or the immobilization matrix to subtract non-specific binders. 2. Specialized SELEX for Complex Proteins: Membrane Protein SELEX: For receptors and channels. Requires special handling (e.g., use of nanodiscs, detergent micelles, or whole cells overexpressing the target). Post-Translationally Modified Protein SELEX: For targets where phosphorylation, glycosylation, etc., are essential for function. 3. Cell-SELEX (for Cell-Surface…
What is an Aptamer? An aptamer is a short, single-stranded DNA or RNA oligonucleotide that folds into a specific 3D structure, allowing it to bind to a target molecule (like a cytokine) with high affinity and specificity, akin to a monoclonal antibody. Why Target Cytokines with Aptamers? Cytokines are key signaling proteins in immune and inflammatory responses. Dysregulation is implicated in diseases like: Autoimmune disorders: Rheumatoid arthritis, psoriasis, inflammatory bowel disease. Cancer: Tumor microenvironment signaling. Cytokine Storms: Severe COVID-19, sepsis. Neurological diseases. Aptamers offer advantages over traditional antibody-based therapies: High Specificity: Can distinguish between closely related cytokine isoforms or conformational states. Controlled Synthesis: Chemically produced, no batch-to-batch variation. Modifiability: Easily conjugated with drugs, fluorophores, or nanoparticles. Low Immunogenicity: Less likely to cause an immune response. Stability: Generally more stable than proteins. The Aptamer Screening Service Workflow (SELEX) A professional service will manage the entire SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process. Here’s a typical pipeline: Phase 1: Project Design & Target Preparation Consultation: Define the goal—neutralization, detection, or delivery. Target Selection: Which cytokine? (e.g., TNF-α, IL-6, IL-1β, IFN-γ). Requires a high-purity, bioactive protein. Services often help with recombinant expression/purification if needed. Library Design: A vast random-sequence oligonucleotide library (10^14-10^15 unique sequences) is the starting point. Libraries can be DNA, RNA, or contain modified…
Why Target Protein Kinases with Aptamers? Protein kinases are a large family of enzymes that regulate almost all cellular processes by phosphorylating target proteins. Their dysregulation is a hallmark of many diseases, especially cancer, making them prime therapeutic targets. Advantages of Aptamers over Traditional Kinase Inhibitors: High Specificity: Can be selected to distinguish between highly conserved kinase family members or even between active/inactive conformations. Modifiable Chemistry: Easy chemical modification for stability (e.g., 2'-F, 2'-O-methyl) and labeling (e.g., fluorophores, biotin). Non-Immunogenic: Unlike antibodies, they are chemically synthesized, reducing batch-to-batch variability. Reversible Inhibition: Typically act as competitive inhibitors, which can be desirable for certain therapeutic strategies. Cell-Permeable Versions: Spiegelmers (L-aptamers) or nanoparticle conjugation can enable intracellular targeting. Core Screening Service Workflow (SELEX) The service revolves around SELEX (Systematic Evolution of Ligands by EXponential Enrichment), specifically optimized for kinases. 1. Project Design & Library Selection: Target Definition: Which kinase? Which conformation (active, inactive, substrate-bound)? Which domain (catalytic, regulatory)? Library Design: Standard DNA/RNA libraries or modified (e.g., 2'-F pyrimidines for nuclease resistance). Library diversity is typically >10^14 unique sequences. 2. Target Preparation: Protein Quality is Critical: The kinase must be highly pure, correctly folded, and functional. Services often use recombinant kinases with tags (GST, His) for immobilization. Immobilization Strategy: Crucial step. Common methods include: Biotin-Streptavidin: Biotinylated…
What is an Aptamer? An aptamer is a short, single-stranded DNA or RNA oligonucleotide that binds to a specific target molecule (like a protein) with high affinity and specificity. They are often called "chemical antibodies" but offer advantages like smaller size, chemical stability, and in-vitro generation. The Core Service: SELEX (Systematic Evolution of Ligands by EXponential Enrichment) The standard method for aptamer screening is SELEX. A specialized service will manage this entire iterative, high-complexity process for you. General SELEX Workflow: Target Preparation & Immobilization: Your service provider will prepare your purified protein. It is often immobilized on a solid support (beads, column, plate) to separate bound from unbound sequences. Incubation with Library: A vast, random synthetic oligonucleotide library (10^13 - 10^15 unique sequences) is incubated with the target. Partitioning: Weak or non-binding sequences are washed away. Tightly bound aptamers are retained. Elution & Amplification: The bound sequences are eluted and amplified by PCR (for DNA) or RT-PCR (for RNA). Stringency & Counter-SELEX: Subsequent rounds introduce increased washing stringency and incubation with non-target molecules (e.g., similar proteins, immobilization matrix) to filter out non-specific binders. This is crucial for specificity. Cloning & Sequencing: After 8-15 rounds, the enriched pool is cloned and sequenced to identify individual candidate aptamers. Characterization &…
Aptamer Screening via HT-SELEX (High-Throughput Systematic Evolution of Ligands by Exponential Enrichment) is the modern, powerful method for discovering aptamers. Let's break down what this service entails, its process, advantages, and key considerations. What is an Aptamer? First, a quick reminder: Aptamers are single-stranded DNA or RNA oligonucleotides that bind to a specific target molecule (proteins, small molecules, cells, viruses) with high affinity and specificity, analogous to antibodies. They are often called "chemical antibodies." What is HT-SELEX? Traditional SELEX is iterative and low-throughput. HT-SELEX supercharges this process by integrating: Next-Generation Sequencing (NGS): To analyze the entire aptamer pool at each round. Advanced Bioinformatics: To identify binding motifs and track enrichment. Automation: Using robotics for partitioning (e.g., magnetic beads, microfluidics) to increase throughput and reproducibility. This results in a faster, more efficient, and data-driven screening process. Standard HT-SELEX Service Workflow A typical service provider will follow these steps: 1. Project Design & Library Synthesis Target Preparation: You provide the target (recombinant protein, small molecule conjugate, whole cell, etc.). Its purity and stability are critical. Library Design: A randomized oligonucleotide library is synthesized (typically 10^14 - 10^15 unique sequences). Libraries can be DNA, RNA, or modified nucleotides (e.g., SOMAmers) for enhanced stability and affinity. 2. The Selection Rounds (Cycles of…
Core Concept of NGS-SELEX Traditional SELEX uses a few rounds of selection and cloning/Sanger sequencing of a handful of clones. NGS-SELEX performs deep sequencing (millions to billions of reads) at every selection round. This allows you to: Track the entire evolution of the oligonucleotide pool in real-time. Identify enriched sequences and families early. Perform sophisticated bioinformatics analysis to find winners, not just rely on final round abundance. Dramatically reduce the number of selection rounds needed (often 3-6 rounds instead of 8-15). Standard Service Workflow A full-service provider would typically offer the following pipeline: 1. Project Design & Library Synthesis Consultation: Target properties (protein, small molecule, cell), desired aptamer properties (Kd, specificity, buffer conditions). Library Design: Standard (40-60 nt random region) or custom (doped libraries, modified nucleotides like 2'-F, 2'-OMe, SOMAmers). Primer & Library Synthesis: Providing the initial, highly diverse DNA or RNA library (10^14 - 10^15 unique sequences). 2. SELEX Selection Immobilization: Immobilizing the target (on beads, column, plate) or using solution-based techniques (capture-SELEX, toggle-SELEX). Counter-Selection: Including steps to remove binders to immobilization matrix or off-targets. Stringency Control: Increasing selection pressure over rounds (e.g., reduced target concentration, increased wash stringency). Amplification: Careful PCR (with optimization to minimize bias) to regenerate the pool for the next round. 3. NGS & Core Bioinformatics Sample Preparation: Preparing sequencing…
What is Subtractive SELEX? It is a specialized version of SELEX used to generate aptamers (single-stranded DNA or RNA oligonucleotides) that bind with high affinity and specificity to a target of interest (e.g., a protein, cell, small molecule) while actively excluding binding to closely related non-targets (e.g., a non-pathogenic vs. pathogenic strain, a healthy vs. cancerous cell, or a target in a complex mixture). The "subtractive" step removes sequences that bind to unwanted counter-targets, ensuring the final aptamer pool is highly specific. Core Workflow of a Subtractive SELEX Service A typical service follows these key stages: 1. Project Design & Library Synthesis Client Consultation: Defining the target of interest (e.g., recombinant protein, whole cell) and the critical counter-target(s) for subtraction (e.g., isotype control protein, non-target cell line). Library Design: A service provider synthesizes a vast random-sequence oligonucleotide library (typically 10^14 - 10^15 unique sequences) flanked by constant primer regions. 2. The Subtractive SELEX Cycle (Repeated 8-15 Rounds) This is the iterative heart of the service: * a. Negative Selection (Subtraction): The oligonucleotide pool is incubated with the counter-target (or complex background, like serum). Sequences that bind to this unwanted material are discarded. * b. Positive Selection: The unbound sequences from (a) are then incubated with the target of interest. The bound sequences are recovered. * c. Washing: Non-specific or weakly bound sequences are washed away.…
