Aptamers are short, single-stranded oligonucleotides (DNA or RNA) that bind to specific targets (proteins, small molecules, cells) with high affinity and specificity. Their generation has traditionally been dominated by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. However, this method has significant limitations, driving innovation toward non-SELEX approaches. 1. Current Predominant Method: SELEX SELEX is an iterative, in vitro selection-amplification process with several variations, each suited for different targets and applications. Core Steps of SELEX: Library Creation: A random oligonucleotide library (10¹³–10¹⁵ sequences) flanked by fixed primer sites. Incubation & Binding: Library exposed to the target. Partitioning: Separation of target-bound sequences from unbound ones. Amplification: PCR (DNA) or RT-PCR (RNA) of bound sequences. Repetition: Typically 8–15 cycles to enrich high-affinity binders. Cloning & Sequencing: Identification of enriched aptamer candidates. Key Variations of SELEX: Cell-SELEX: Uses whole cells as targets, ideal for biomarker discovery. Capture-SELEX: For small molecules, using immobilized targets. Capillary Electrophoresis-SELEX (CE-SELEX): High-efficiency partitioning via electrophoresis. Microfluidic-SELEX: Reduces time and reagent use via automation. Magnetic Bead-Based SELEX: Easy separation using target-coated beads. Limitations of SELEX: Time-Consuming: Multiple cycles take weeks to months. Labor-Intensive: Requires significant hands-on effort. Amplification Bias: PCR can favor certain sequences unrelated to binding. Limited Sequence Diversity: Early high-affinity binders can dominate, reducing diversity. Cost: High reagent and time costs. 2. Emerging Trend: Non-SELEX Approaches…
Excellent choice! Aptamer Screening is the core process for discovering these synthetic, single-stranded DNA or RNA molecules that bind to a specific target with high affinity and specificity. It's often called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Here’s a comprehensive breakdown of aptamer screening, from concept to modern advancements. 1. The Core Principle: SELEX The traditional screening method is an in vitro Darwinian evolutionary process. The basic cycle is repeated until a pool of high-affinity aptamers is obtained. Key Steps: Library Design: Start with a vast random-sequence oligonucleotide library (10^13 - 10^15 different molecules). Each molecule has a central random region (20-60 nucleotides) flanked by constant primer regions for PCR amplification. Incubation: The library is incubated with the target molecule (e.g., a protein, small molecule, cell). Partitioning: Unbound sequences are washed away. Bound sequences (potential aptamers) are retained. This is the most critical step, dictating the success of the entire screen. Elution: The bound sequences are recovered (e.g., by heating, denaturing agents, or target digestion). Amplification: The recovered sequences are amplified by PCR (for DNA) or RT-PCR (for RNA) to create an enriched pool for the next round. Iteration: Steps 2-5 are repeated (typically 5-15 rounds) under increasingly stringent conditions (e.g., shorter incubation time, more washes, competitive agents) to select…
