“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…
What is the Core Service? The service provider uses an iterative, in vitro selection process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to screen vast random oligonucleotide libraries (10^14 - 10^15 unique sequences) against your target protein. The output is a set of characterized aptamer sequences that bind to the viral capsid. Standardized Screening Workflow A professional service will manage this entire pipeline: 1. Project Design & Target Preparation: Target Discussion: Defining the specific capsid protein (e.g., HIV-1 CA, HBV core, SARS-CoV-2 N), its form (full-length, domain, assembled capsid/nucleocapsid), and purity. Target Immobilization: The protein is often immobilized on a solid support (beads, plate) to facilitate separation of bound/unbound sequences. Some services offer solution-phase or capillary electrophoresis (CE-SELEX) methods for higher stringency. 2. SELEX Selection Rounds (Cycles 5-15): Incubation: The oligonucleotide library is incubated with the target. Partitioning: Unbound sequences are washed away; bound sequences are retained. Elution: Bound aptamers are eluted (e.g., by heating, denaturing agents). Amplification: Eluted aptamers are amplified by PCR (for DNA) or RT-PCR (for RNA). Purification: The amplified pool is purified for the next selection round. Counter-Selection: To ensure specificity, the pool is often passed through a negative control (e.g., irrelevant protein, cell lysate) to remove non-specific binders. 3. Sequencing & Identification: High-Throughput…
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.…
What is Whole-cell SELEX? Whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a technique used to discover aptamers (single-stranded DNA or RNA molecules) that bind specifically to a target living cell. Unlike traditional SELEX that uses a purified protein target, whole-cell SELEX presents the target in its native, complex cellular environment. This allows for the selection of aptamers against: Native cell-surface proteins in their proper folding and post-translational modifications. Complex targets like transmembrane receptors in their natural lipid environment. Unknown surface biomarkers without prior knowledge of the cell's molecular makeup. Specific cell states (e.g., activated, cancerous, infected) based on differences in surface expression. The Core Process: How Whole-cell SELEX Works A professional service will manage this complex, iterative pipeline: Library & Design: Starting with a vast, random synthetic oligonucleotide library (10^14 - 10^15 unique sequences). Positive Selection: Incubating the library with the target cells (e.g., cancer cells, stem cells, bacteria). Aptamers that bind to any surface structure are retained. Counter-Selection (Critical Step): The bound pool is then exposed to non-target or control cells (e.g., healthy cells, a different cell line). Sequences that bind to these are discarded. This step is crucial for generating specificity. Elution & Amplification: Aptamers specifically bound to the target cells are recovered, amplified by PCR…
Traditional SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a method to select high-affinity, specific nucleic acid aptamers from a vast random library (10¹³-10¹⁵ sequences). The bottleneck has always been the final cloning and Sanger sequencing of only a few dozen candidates, which often misses rare, high-performance aptamers. NGS-assisted SELEX integrates Next-Generation Sequencing at multiple rounds of the SELEX process. This provides a massive, data-rich view of the entire evolutionary landscape, enabling intelligent selection and identification of the best aptamers. Typical Workflow of an NGS-Assisted SELEX Service A professional service provider will manage this entire pipeline: Project Design & Library Synthesis: Collaboration to define target (protein, small molecule, cell), counter-selection requirements, and library design (random region length, fixed primers for NGS). Parallel SELEX Execution: Performing the iterative selection process (binding, partitioning, amplification) across multiple rounds (usually 8-12). Key NGS Integration Points: Initial Library Analysis: Sequencing the naive library to confirm diversity and complexity. Monitoring Rounds (e.g., Rounds 3, 6, 9): Taking small samples from intermediate rounds for NGS. This is the critical advantage. It tracks: Sequence Enrichment: Which families are becoming more abundant. Diversity Collapse: When to stop selection before losing good candidates. Informed Decision-Making: Data guides adjustments in selection stringency for subsequent rounds. Final Round Deep Sequencing: Comprehensive NGS of…
