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 Screening Service? It is a contract-based service where a specialized laboratory uses Systematic Evolution of Ligands by EXponential enrichment (SELEX) to discover single-stranded DNA or RNA molecules (aptamers) that bind with high affinity and specificity to your target molecule (e.g., a protein, an antibody's constant region, or a cell-surface receptor). Core Service Components A full-service provider typically offers an end-to-end pipeline: 1. Project Design & Target Preparation Consultation: Defining the goal (e.g., detection, inhibition, delivery). Target Characterization: Ensuring the target (purified protein, antibody, receptor-expressing cells) is properly formatted and validated. Negative Selection/Counter-SELEX: Designing the screening to avoid binders to similar, non-target structures (e.g., the Fc region of a different antibody isotype, a common cell surface protein). 2. Library & Selection (The Core SELEX Process) Library Design: Using a diverse random oligonucleotide library (typically 10^14 - 10^15 unique sequences). Selection Method: The choice of method is critical and depends on the target: Protein SELEX: For purified, soluble targets immobilized on beads or in solution. Cell-SELEX: For membrane receptors in their native conformation on live cells. Excellent for discovering aptamers to unknown receptor complexes. Capture-SELEX/Toggle-SELEX: For difficult-to-immobilize targets or to increase stringency. In Vivo SELEX: For discovering aptamers that home to specific tissues in vivo. Iterative Rounds: Typically 8-15 rounds of…
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 &…
Core Concept: What is SELEX? SELEX (Systematic Evolution of Ligands by EXponential Enrichment) is an iterative, in vitro selection process. It starts with a vast, random library of oligonucleotides (10^14 - 10^15 unique sequences) and, over multiple rounds, enriches for those that bind to the target. Standard Multi-Round SELEX Screening Service Workflow A full-service provider will typically manage the entire process, which can be broken down into key phases: Phase 1: Project Design & Target Preparation Target Consultation: Defining the target (e.g., protein, small molecule, cell, virus). Critical discussion of target purity, immobilization strategy, and selection conditions (buffer, temperature, counter-selection). Library Design: Selection of a random library (e.g., 40-nt random core with fixed primer sites). Options include DNA, RNA (requiring reverse transcription), or modified libraries (e.g., with 2'-F pyrimidines for nuclease resistance). Immobilization Strategy: The service provider will choose the best method: Immobilized Target: (Most common for proteins) Binding target to beads (streptavidin, Ni-NTA for His-tag) or columns. Counter-Selection: Using negative control surfaces (e.g., blank beads, related but undesired proteins) to subtract non-specific binders. Phase 2: The SELEX Cycle (Repeated 8-15 Rounds) This is the core iterative screening process. Each round consists of: Incubation: The oligonucleotide library is incubated with the target under defined conditions. Partitioning: Separation of…
What is Counter-SELEX? First, a quick recap of SELEX (Systematic Evolution of Ligands by EXponential Enrichment): SELEX is an iterative process to isolate specific DNA or RNA aptamers from a vast random library (10^14 - 10^15 sequences) that bind tightly to a target molecule (e.g., a protein, small molecule, cell). Counter-SELEX is a powerful refinement to this process. Its core purpose is to improve specificity by negative selection. How it works: During or between rounds of positive selection (binding to the desired target), the oligonucleotide pool is exposed to one or more counter-targets. The Goal: Sequences that bind to these counter-targets are deliberately removed or depleted from the pool. Only sequences that bind specifically to the desired target and not to the closely related counter-targets are carried forward. Common Counter-Targets: Structural analogs: For a small-molecule drug, you might use its inactive metabolite or a similar drug from the same class. Protein isoforms or family members: To develop an aptamer for a specific kinase, you'd use other kinases from the same family as counter-targets. Immobilization matrix: If the target is immobilized on beads, pre-incubating the library with "blank" beads removes matrix binders. Related cell types: For a cell-specific aptamer (e.g., cancer vs. healthy), the healthy cells are used as the counter-target. What Does a…
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.…
Toggle-SELEX is a sophisticated and powerful variant of the traditional SELEX process for aptamer development, specifically designed to generate aptamers that recognize multiple, closely related targets or a specific epitope common across different species/conditions. Let's break down what an Aptamer Screening Service using Toggle-SELEX entails, its applications, and what you should consider when selecting a service provider. What is Toggle-SELEX? The core idea of Toggle-SELEX is to "toggle" or alternate the selection pressure between two (or more) related target molecules during the SELEX rounds. Traditional SELEX: Uses a single target to evolve aptamers with high affinity for that specific target. It often negatively selects against related molecules (counter-selection) to ensure specificity. Toggle-SELEX: Actively uses two positive selection targets in an alternating pattern. For example: Round 1: Select against Target A (e.g., human protein). Round 2: Select against Target B (e.g., mouse ortholog of the same protein). Round 3: Back to Target A, and so on. Counter-selection against unrelated structures is still used to maintain general specificity. This process enriches for nucleic acid sequences that bind to a conserved structural epitope present on both targets, while sequences that bind to unique epitopes on only one target are filtered out. Key Applications of Toggle-SELEX This method is invaluable when you need cross-reactive or broad-spectrum recognition: Cross-Species Reactive Aptamers: Develop aptamers for preclinical research. For example, an…
What is SELEX? First, a quick recap: SELEX (Systematic Evolution of Ligands by EXponential enrichment) is the gold-standard process for discovering aptamers (single-stranded DNA or RNA molecules that bind to a specific target with high affinity and specificity). It involves iterative cycles of binding, partitioning, and amplification. "Free Solution SELEX" Explained Free Solution SELEX (also known as non-immobilized SELEX) is a specific technical approach. Its key characteristic is that neither the target molecule nor the library is fixed to a solid support (like beads, a column, or a chip) during the binding step. How it works: Binding: The random oligonucleotide library is mixed with the free, soluble target in solution. Partitioning: The key challenge is separating the bound sequences from the unbound ones without using immobilization. Common methods include: Nitrocellulose Filter Binding: Aptamer-target complexes are trapped on a filter, while free sequences pass through. Gel Filtration/Size Exclusion: Separates complexes (larger) from unbound sequences (smaller). Capture Techniques: Using a brief, weak tag on the target (like biotin) to pull down complexes after binding in solution. Amplification: The bound sequences are eluted, amplified by PCR (for DNA) or RT-PCR (for RNA), and purified for the next round. Advantages of Free Solution SELEX: Native Target Conformation: The target is in its natural, free state. There's no risk of…
What is CE-SELEX? SELEX (Systematic Evolution of Ligands by EXponential Enrichment) is the standard process for aptamer development. It involves iterative rounds of selection and amplification to enrich nucleic acid sequences that bind tightly to a target molecule. Traditional SELEX often uses immobilization of the target on beads or filters, which can be slow (8-15 rounds) and may introduce bias by selecting for sequences that bind to the immobilization matrix itself. CE-SELEX uses Capillary Electrophoresis as the separation mechanism. The key principle is that when an aptamer binds to its target, it forms a complex with a different charge-to-size ratio, causing it to migrate at a different time (shifted peak) in the capillary compared to the unbound nucleic acid library. This complex can be isolated and collected with exquisite precision. Core Advantages of a CE-SELEX Screening Service A service provider offering CE-SELEX delivers significant benefits: Extreme Speed and Efficiency: Often requires only 2-4 rounds of selection to obtain high-affinity aptamers (nanomolar to picomolar Kd), compared to many more rounds in traditional SELEX. This translates to weeks or months of time saved. Solution-Phase Selection: The target is free in solution, eliminating immobilization bias. This allows for selection against targets in their native conformation and enables selection for small molecules and…
Core Principle Nitrocellulose membrane filter binding exploits a simple but powerful property: nitrocellulose avidly binds proteins and protein-nucleic acid complexes, but does not efficiently bind free, single-stranded DNA or RNA. By passing a mixture of the target protein and a random oligonucleotide library through the membrane, sequences that bind to the protein are retained (as a complex), while unbound sequences are washed away. Typical Workflow of a Service Provider A professional service will manage this complex, iterative process for you: 1. Project Design & Library Synthesis Consultation: Defining your target (purified protein is essential), desired aptamer properties (affinity, specificity, buffer conditions), and format (DNA or RNA). Library Design: A synthetic library of up to 10^15 random sequences (e.g., 40-60 nt random core, flanked by constant primer regions) is prepared. 2. The SELEX Cycles (Iterative Screening) Incubation: The target protein is incubated with the nucleic acid library under optimized conditions (buffer, temperature, time). Positive Selection (Binding & Capture): The mixture is passed through a nitrocellulose membrane. Protein-aptamer complexes stick to the membrane. Washing: Mild washing removes weakly bound or non-specific sequences. Elution: Bound sequences are recovered by denaturing the protein (e.g., using heat, phenol-chloroform, or high-concentration urea). Amplification: For DNA SELEX: The eluted DNA is directly amplified by PCR. For…
