Technical Highlights of the Aptamer Development Process Aptamer development is centered around the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. Its key technological highlights can be summarized in the following stages: 1. Library Design & Synthesis Vast Diversity: Creation of a synthetic random-sequence oligonucleotide library (typically 10^14 - 10^15 different sequences) flanked by fixed primer regions. Chemical Modification: Incorporation of modified nucleotides (e.g., 2'-F, 2'-O-Me) to enhance nuclease resistance and binding affinity post-selection. 2. Target-Specific Selection (The Core of SELEX) Incubation: The library is incubated with the immobilized or soluble target molecule (protein, small molecule, cell, etc.). Partitioning: Efficient separation of target-bound sequences from unbound ones using methods like filtration, affinity chromatography, or capillary electrophoresis. Counter-Selection: A critical step to subtract sequences that bind to non-target components (e.g., immobilization matrix or related off-targets), drastically improving specificity. 3. Amplification & Iteration PCR/RT-PCR: Recovery and exponential amplification of the bound sequences to generate an enriched pool for the next selection round. Stringency Control: Gradual increase in selection pressure (e.g., reduced target concentration, shorter incubation time, stringent washes) over successive rounds (typically 8-15 rounds) to drive the evolution of high-affinity, specific binders. 4. Cloning, Sequencing & Characterization Clone Isolation: Post-SELEX, the enriched pool is cloned, and individual sequences are identified. Bioinformatics…
Core Concept: SELEX The synthesis of aptamers is not a direct chemical construction from a blueprint, but rather a selection and amplification process from a vast random library. The primary method is called SELEX (Systematic Evolution of Ligands by EXponential enrichment). The Technical Process: A Step-by-Step Breakdown The entire process can be divided into three major phases: Library Design & Synthesis, Selection (SELEX), and Post-SELEX Optimization & Production. Phase 1: Library Design and Chemical Synthesis This is the starting raw material. Design: A single-stranded DNA (ssDNA) or RNA library is designed with a central randomized region (typically 20-60 nucleotides) flanked by constant primer regions. Random Region: Provides the vast sequence diversity (e.g., 40 random positions = 4⁴⁰ ~ 1.2x10²⁴ possible sequences). Constant Regions: Essential for PCR amplification during later steps. Chemical Synthesis: The DNA library is synthesized using solid-phase phosphoramidite chemistry (the same as for oligonucleotide drugs and primers). Machines introduce nucleotides one-by-one in a controlled cycle (Deprotection, Coupling, Capping, Oxidation). For RNA libraries, transcription from a DNA template is used, or they are synthesized directly with 2'-OH protecting groups. Phase 2: The SELEX Cycle (Iterative Selection & Amplification) This is the iterative engine that finds the needle (high-affinity aptamers) in the haystack (random library). For ssDNA SELEX: Key Technical Steps in Detail: Incubation: The synthetic library…
Introduction to Nucleic Acid Aptamers What Are Aptamers? Nucleic acid aptamers are short, single-stranded DNA or RNA molecules that fold into specific three-dimensional structures, enabling them to bind to target molecules with high affinity and specificity. The word "aptamer" comes from the Latin aptus (to fit) and the Greek meros (part). Key Characteristics Size: Typically 20-80 nucleotides in length Binding affinity: Often in nanomolar to picomolar range Specificity: Can distinguish between closely related molecules Synthetic origin: Created through in vitro selection processes How Aptamers Are Created: SELEX Aptamers are developed through SELEX (Systematic Evolution of Ligands by EXponential enrichment), an iterative process involving: Incubation of a random nucleic acid library with the target Separation of binding sequences from non-binders Amplification of selected sequences Repetition over multiple rounds (typically 8-15 cycles) Advantages of Aptamers Compared to antibodies, aptamers offer: Chemical synthesis: Reproducible production without batch variability Modifiability: Can be chemically modified for stability and functionality Temperature stability: Can often be refolded after denaturation Non-immunogenic: Low immunogenicity in therapeutic applications Target range: Can bind to toxins, non-immunogenic molecules, and small compounds Applications Diagnostics: Biosensors, detection assays (aptasensors) Therapeutics: Targeted drug delivery, direct inhibitors ("chemical antibodies") Research tools: Protein detection, cellular imaging, biomarker discovery Analytical chemistry: Affinity purification, chromatography…
Excellent question. It's important to clarify terminology first: while aptamers are often selected against targets we classically call "antigens" (e.g., proteins on pathogens), the term "antigen" (antibody-generator) is specific to the immune system. In aptamer selection (SELEX), the target is more accurately called the "target molecule" or "ligand." However, your question is about the types of targets used for aptamer selection. These targets can range from small molecules to whole cells. The choice of target type dictates the selection strategy (e.g., purified protein SELEX vs. Cell-SELEX). Here’s a comprehensive breakdown of antigen/target types for aptamer selection: 1. Proteins (The Most Common Category) This is the largest class of targets, mimicking the traditional antigen space for antibodies. Membrane Proteins: Receptor tyrosine kinases (EGFR, VEGFR), G-protein-coupled receptors (GPCRs), ion channels, transporters. Crucial for cell-surface targeting. Soluble Proteins: Cytokines (TNF-α, IFN-γ), growth factors (VEGF), hormones (insulin), enzymes (thrombin), antibodies themselves, viral coat proteins (SARS-CoV-2 Spike protein). Post-Translationally Modified Proteins: Phosphorylated proteins (for signaling studies), glycoproteins (like PSA). Protein Domains or Epitopes: A specific folded region or a short linear epitope of a larger protein. 2. Whole Cells (Cell-SELEX) A powerful method to generate aptamers for unknown cell-surface biomarkers, often for cancer or stem cell targeting. Target Cells: Cancer cell lines, primary tumor cells, bacteria, viruses,…
What is SELEX? SELEX (Systematic Evolution of Ligands by EXponential enrichment) is an in vitro combinatorial chemistry technique used to isolate high-affinity, high-specificity nucleic acid ligands (aptamers) from a vast random-sequence library against a target molecule. Think of it as "molecular evolution in a test tube." Starting with a pool of ~10¹³-10¹⁵ random sequences, SELEX uses iterative cycles of selection and amplification to "evolve" the few molecules that bind best to the target, much like natural selection evolves organisms. Core Principle The principle is based on three repeating steps: Incubation: A vast library of random oligonucleotides is exposed to the target. Partitioning: The rare molecules that bind to the target are separated from the non-binders. Amplification: The bound sequences are amplified (usually by PCR for DNA or RT-PCR for RNA) to create an enriched pool for the next selection round. After 8-20 rounds, the pool becomes dominated by sequences with high binding affinity and specificity for the target. The Standard SELEX Process (Step-by-Step) 1. Library Synthesis: A synthetic library contains 10¹³ to 10¹⁵ different single-stranded DNA or RNA molecules. Each molecule has a central random region (20-80 nucleotides) flanked by constant primer regions for amplification. 2. Incubation & Binding: The library is incubated with the target molecule (e.g., a protein,…
Nucleic Acid Aptamer Libraries Screening: an Efficient Method for Discovering Specific Binding Molecules Aptamers are oligonucleotide sequences (DNA or RNA) consisting of 20-110 nucleotides. Aptamers have high molecular recognition capabilities for different types of targets such as nucleic acids, proteins, cells, and some small molecules, can distinguish subtle differences, and have high affinity and specificity, and are easy to chemically modify. Aptamers are usually oligonucleotide fragments obtained from nucleic acid molecule libraries using in vitro screening technology, namely systematic evolution of ligands by exponential enrichment (SELEX). The structure and function of aptamers are similar to antibodies, but they are smaller in size, have shorter production time, lower cost, and are easier to synthesize. Based on the characteristics of aptamers that can specifically bind to a variety of target molecules, they are widely used in food safety, environmental monitoring, biomedical research and other fields. Aptamers can be used in drug development to design drugs that target specific molecules; many aptamers are used to make biosensors that can diagnose some specific infectious diseases; aptamers can be used as carriers to deliver genes to specific cells or tissues, and have certain application prospects in gene therapy. Since nucleic acid aptamers can be prepared…
Aptamer Screening for Small Molecule Service Process
Categorization & Core Principle All these techniques share the common goal of SELEX (Systematic Evolution of Ligands by EXponential enrichment): isolating high-affinity aptamers from a vast random-sequence library (10^14 - 10^15 sequences) through iterative cycles of Binding → Separation → Amplification → Purification. Your listed techniques primarily differ in the separation method used to partition target-binding sequences from non-binders. Detailed Analysis & Context A. Classical SELEX (Solution-based) What it is: The foundational, versatile protocol. The target is often immobilized to facilitate separation. Separation Method: Filtration, affinity columns, or magnetic beads (if target is tagged/bound to a bead). Note: "SELEX technology" is the umbrella term. Techniques B, C, D, and E are all variants of SELEX that use different separation principles. B. Solid-Phase SELEX (A specific implementation of classical SELEX) Clarification: This is the most common practical implementation for small molecules. The "solid phase carrier" is typically streptavidin-coated beads if the target is biotinylated, or an activated resin if the target is chemically immobilized. Key Advantage: Excellent for counter-selection (to remove sequences that bind to the solid support or similar non-target molecules). C. Centrifugal Precipitation / Particle-Based SELEX Best For: As you noted, it's ideal for cells, bacteria, or large vesicles. For true small molecules, this method is less common unless the small molecule is…
Core Challenge with Small Molecules Small molecules (e.g., drugs, toxins, metabolites, <1000 Da) lack the large, multi-epitope surfaces of proteins. This makes traditional selection methods difficult because: Immobilization: Hard to attach to a solid phase without masking the target area. Limited Binding Interfaces: Offer fewer points for oligonucleotide interaction. Low Signal-to-Noise: Distinguishing specific binders from non-specific binders is tougher. Key Aptamer Screening Techniques Used for Small Molecules KMD Bioscience likely employs a combination of these advanced SELEX (Systematic Evolution of Ligands by EXponential Enrichment) variants: 1. Capture-SELEX (The most common for small molecules) Principle: Instead of immobilizing the small molecule, a DNA library with a fixed primer sequence is immobilized on beads. The small molecule is free in solution. Process: The small molecule is introduced. Sequences that bind to it undergo a conformational change, freeing them from the bead into the solution. These eluted sequences are then amplified. Advantage: The target remains in its native, unmodified state, preserving its structure and function. Ideal for targets that are difficult to tag or immobilize. 2. Graphene Oxide-SELEX (GO-SELEX) Principle: Utilizes graphene oxide's ability to adsorb single-stranded DNA (ssDNA) non-specifically via π-π stacking. Process: The ssDNA library is incubated with GO. Unbound sequences are discarded. The small molecule target is then added. Aptamers…
Aptamer Selection Small molecule aptamer screening techniques generally include the following steps: A. Initial library design: Build a DNA, RNA or peptide library containing a large number of random sequences that have the potential to bind to small target molecules. B. Screening process: The main steps include target binding to the library, isolation and purification of the binding target molecule aptamer PCR amplification, iterative screening and so on. After each round of screening, the screening results can be verified and analyzed through technologies such as sequencing. C. Optimization and validation: The selected aptamers were optimized to improve their affinity, specificity and stability. Subsequently, a series of experiments were carried out to verify its binding ability and application effect. KMD Bioscience Aptamer Screening Service: A Comprehensive Workflow Your description accurately captures the three universal pillars of in vitro selection (SELEX). KMD Bioscience would operationalize these into a robust, client-tailored service. A. Initial Library Design & Preparation Library Diversity: Construction of high-complexity synthetic libraries (typically 10^14 - 10^15 unique sequences) containing a central random region (e.g., 30-60 nucleotides) flanked by constant primer regions. Format Flexibility: Offering DNA, RNA, or modified nucleotide (e.g., 2'-F, 2'-O-Me) libraries to balance stability, cost, and affinity requirements. Target Immobilization: A critical initial step where the small molecule target is…
