Aptamers are synthetic, single-stranded oligonucleotides (DNA or RNA) that fold into specific three-dimensional shapes, allowing them to bind to target molecules with high affinity and specificity. Often called "chemical antibodies," they are identified through an in vitro selection process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Here are their key characteristics: 1. High Specificity and Affinity They can distinguish between targets with subtle differences (e.g., between two proteins differing by a few amino acids, or between chiral molecules). Binding affinities (K_d) can reach the nanomolar to picomolar range, comparable to antibodies. 2. Versatile Target Range Target virtually any class of molecule: proteins, peptides, small molecules, ions, whole cells, viruses, and even toxins. 3. Synthetic Origin & In Vitro Selection Produced entirely in vitro via SELEX, avoiding animal use. Selection conditions can be precisely controlled to obtain aptamers with desired properties (e.g., stability in specific pH or temperature). 4. Small Size Typically 20–80 nucleotides long (6–25 kDa), much smaller than antibodies (~150 kDa). Allows better tissue penetration and access to cryptic epitopes. 5. Excellent Stability Thermal stability: Can be renatured after denaturation. Chemical stability: Generally more robust than proteins. DNA aptamers are especially stable for long-term storage. Modifiable: Can be chemically synthesized with modifications (e.g., 2'-fluoro, 2'-O-methyl, PEGylation) to enhance nuclease resistance and pharmacokinetics. 6. Low Immunogenicity Being composed…
What are Aptamers? Aptamers are single-stranded DNA or RNA oligonucleotides that fold into specific 3D shapes, enabling them to bind with high affinity and specificity to a target molecule (e.g., proteins, small molecules, cells, viruses). They are often termed "chemical antibodies." The process to discover them is called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The Core Principle: SELEX SELEX is an iterative, in vitro evolutionary process that mimics natural selection. It starts with a vast random library (10¹³–10¹⁵ unique sequences) and enriches those that bind to the target over multiple rounds. Key Steps in a Single SELEX Cycle: Library Preparation: A synthetic oligonucleotide library is created with a central random region (20–60 nucleotides) flanked by fixed primer-binding sites for PCR amplification. Incubation (Binding): The library is incubated with the target under controlled conditions (buffer, temperature, time). A portion of the diverse sequences will bind to the target with varying affinity. Separation (Partitioning): This is the most critical step. Bound sequences (the "hits") must be efficiently separated from unbound ones. Methods include: Immobilized Targets: Target is fixed on a column, beads, or filter. Unbound sequences are washed away. Nitrocellulose Filter Binding: For protein targets; protein-nucleic acid complexes are retained. Magnetic Bead Separation: Very common and versatile. Capture-SELEX: For…
SELEX Method for Screening Aptamers: A Comprehensive Guide Overview SELEX (Systematic Evolution of Ligands by EXponential Enrichment) is the foundational in vitro technique for isolating aptamers - single-stranded DNA or RNA oligonucleotides that bind specific targets with high affinity and specificity. Key Concepts Aptamers: "Chemical antibodies" that fold into 3D structures for target binding Targets: Can be proteins, small molecules, cells, viruses, or even entire organisms Library: Typically 10¹³-10¹⁵ random sequences (30-80 nucleotides long) The SELEX Process 1. Library Preparation text Random region (N)ₙ: 30-80 nucleotides Flanked by constant primer regions for PCR amplification DNA libraries: Direct chemical synthesis RNA libraries: DNA template + in vitro transcription 2. Selection Cycle (Repeated 8-15 Rounds) [Target Incubation] → [Partitioning] → [Elution] → [Amplification] → [Conditioning] A. Incubation Library + target molecule in binding buffer Optimized conditions (temperature, ionic strength, pH) B. Partitioning (Critical Step) Separate bound from unbound sequences: Membrane filtration (common for protein targets) Affinity chromatography (immobilized targets) Magnetic separation (bead-conjugated targets) Capillary electrophoresis (high resolution) Microfluidic systems (modern approaches) C. Elution Denature aptamer-target complex Methods: heat, denaturants, or competitive elution D. Amplification DNA aptamers: PCR directly RNA aptamers: RT-PCR → in vitro transcription Counter-selection: Often included to remove non-specific binders E. Conditioning Purify amplified pool for next round Increasing…
What Are Aptamers? Aptamers (from the Latin aptus = "to fit," and Greek meros = "part") are short, single-stranded oligonucleotides (DNA or RNA) that fold into specific 3D shapes, allowing them to bind to a target molecule with high affinity and specificity. They are often called "chemical antibodies" due to their similar function, but they are made of nucleic acids, not proteins. DNA Aptamers Composition: Deoxyribonucleic acid. Structure: Typically more rigid and stable than RNA due to the absence of the 2'-hydroxyl group, which makes it less prone to hydrolysis. Key Features: High Stability: Very resistant to degradation, especially compared to RNA. They are the more robust choice for diagnostic applications outside of controlled environments. Ease of Synthesis: DNA is chemically simpler and cheaper to synthesize and modify at large scales. Simpler Folding: DNA libraries can have less structural diversity than RNA, which may limit the complexity of binding pockets they can form. Common Applications: Biosensors, diagnostic assays, as inhibitors in therapeutics. RNA Aptamers Composition: Ribonucleic acid. Structure: The presence of the 2'-hydroxyl group allows for greater structural diversity and more complex folding (e.g., pseudoknots, tight loops). This often enables higher-affinity binding. Key Features: Structural Complexity: Can form a wider variety of intricate 3D shapes, leading to potentially higher specificity and affinity for challenging targets. Lower…
Aptamers: Structure and Function Definition and Overview Aptamers are short, single-stranded oligonucleotides (DNA or RNA) or peptides that bind to specific target molecules with high affinity and specificity. They are often termed "chemical antibodies" due to their target-binding capabilities, but are entirely synthetic and produced via an in vitro selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Structure of Aptamers 1. Primary Structure Composition: Made of nucleotides (DNA: dA, dT, dG, dC; RNA: A, U, G, C) typically 20-80 bases in length. Sequence: The unique sequence of bases determines the specific three-dimensional shape and thus the binding functionality. 2. Secondary Structure Driven by intramolecular base pairing and stacking, aptamers fold into specific, stable motifs: Stem-loops (Hairpins): Common structural element providing a recognition pocket. G-quadruplexes: Formed by guanine-rich sequences; planar stacks of G-tetrads stabilized by cations (e.g., K⁺). Pseudoknots: Complex structures with nested base-pairing. Bulges and Internal Loops: Provide flexibility and specific interaction points. 3. Tertiary Structure The overall three-dimensional fold, resulting from the arrangement of secondary motifs. This unique 3D shape creates binding pockets, clefts, or surfaces that recognize and bind to the target via: Complementary Shape (Lock-and-Key & Induced Fit): The aptamer folds to match the target's surface topology. Non-Covalent Interactions: Electrostatic interactions, hydrogen bonding, van der Waals forces, and…
Core Principle: SELEX SELEX is an iterative, in vitro process that screens vast random nucleic acid libraries (10^14 - 10^15 sequences) to isolate high-affinity, specific aptamers against a target. Key Screening Methodologies for Liver Cancer The choice of target is paramount and dictates the screening strategy. 1. Cell-SELEX (Whole-Cell SELEX) This is the most common method for discovering aptamers that bind to native cell surface biomarkers without prior knowledge of their identity. Target Cells: Human liver cancer cell lines (e.g., HepG2, SMMC-7721, Huh7, PLC/PRF/5). Counter-Selection Cells: Crucial for specificity. Typically use: Normal human hepatocyte cell lines (e.g., LO2, THLE-3). Non-malignant liver cells or immortalized hepatocytes. Sometimes other cancer cell lines (e.g., from lung, colon) to avoid cross-reactivity. Process: Incubate the ssDNA or RNA library with target liver cancer cells. Wash away unbound sequences. Elute bound sequences (e.g., by heating, trypsinization, or cell lysis). Amplify eluted sequences (PCR for DNA, RT-PCR for RNA). Incubate the enriched pool with counter-selection cells. Sequences that bind are discarded; the unbound pool proceeds. Repeat cycles (usually 8-20 rounds) until a highly enriched pool is obtained. Clone and sequence the final pool for individual aptamer identification. Advantage: Identifies aptamers to unknown, natively folded, and post-translationally modified membrane proteins. 2. Tissue-SELEX…
Screening of Aptamers and Their Potential Application in Targeted Diagnosis and Therapy of Liver Cancer Introduction to Aptamers Aptamers are short, single-stranded DNA or RNA oligonucleotides (typically 20-100 nucleotides) that bind to specific target molecules with high affinity and specificity, similar to antibodies. They are often called "chemical antibodies" but offer advantages including: Ease of synthesis and modification Low immunogenicity Enhanced tissue penetration Thermal stability Lower production costs Screening Methods for Liver Cancer-Specific Aptamers 1. SELEX Technology Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the primary method for aptamer selection: Key adaptations for liver cancer: Cell-SELEX: Using live hepatoma cells (e.g., HepG2, Huh7) as targets Tissue-SELEX: Employing liver cancer tissue specimens In vivo SELEX: Direct screening within animal models Automated SELEX: High-throughput screening platforms 2. Target-Specific SELEX Variations Protein-based SELEX: Against liver cancer biomarkers (AFP, GPC3, etc.) Whole-cell SELEX: For cell surface epitope targeting Toggle SELEX: For cross-reactivity across different liver cancer cell types Applications in Liver Cancer Diagnosis 1. Imaging and Detection Molecular Imaging: Radiolabeled aptamers for PET/CT imaging Fluorescent Aptamers: For intraoperative guidance and tumor margin identification MRI Contrast Agents: Aptamer-conjugated nanoparticles for enhanced imaging 2. Biosensor Development Electrochemical Sensors: For detecting circulating tumor cells Colorimetric Assays: Point-of-care testing for…
Of course. Aptamers, often called "chemical antibodies," are single-stranded DNA or RNA oligonucleotides that bind to specific target molecules (proteins, small molecules, cells, etc.) with high affinity and specificity. Their unique properties—ease of synthesis, stability, low immunogenicity, and small size—make them powerful tools across numerous fields. Here is a comprehensive overview of the key applications of aptamers, categorized by field: 1. Therapeutics & Medicine Aptamers are developed as targeted drugs, often competing with monoclonal antibodies. Targeted Cancer Therapy: Aptamers can deliver toxic drugs, siRNAs, or radioisotopes directly to cancer cells by binding to overexpressed surface markers (e.g., nucleolin, PSMA). This minimizes damage to healthy tissues. Antidotes and Antagonists: The only FDA-approved aptamer to date, Pegaptanib (Macugen), is used for age-related macular degeneration (AMD). It binds and inhibits VEGF-165, a protein promoting abnormal blood vessel growth. Antiviral and Antimicrobial Agents: Aptamers can block viral entry (e.g., against HIV gp120, SARS-CoV-2 spike protein) or inhibit essential bacterial proteins, offering potential alternatives to traditional antibiotics. Anti-inflammatory and Autoimmune Diseases: Aptamers can neutralize inflammatory cytokines (e.g., TNF-α, IL-6) implicated in diseases like rheumatoid arthritis or psoriasis. Targeted Drug Delivery Systems: Aptamers conjugated to nanoparticles or liposomes create "smart" delivery vehicles that release their cargo only at the disease site. 2.…
The Core Principle of SELEX Think of SELEX as artificial selection or directed evolution for molecules. You start with a massive, diverse library of random nucleic acid sequences (typically 10^14 to 10^15 different molecules) and iteratively select for the tiny fraction that binds tightly to your target molecule. The Standard SELEX Process (Step-by-Step) The process is cyclical, typically requiring 5-15 rounds to enrich high-affinity binders. 1. Library Design & Synthesis: A synthetic library is created where each molecule has constant primer regions (for PCR amplification) flanking a central randomized region (20-60 nucleotides long). This creates a "pool of possibility" often called a combinatorial library. 2. Incubation (Binding): The library is incubated with the target molecule (e.g., a protein, small molecule, cell, or even a whole pathogen). Conditions (buffer, temperature, ionic strength) are controlled to favor specific binding. 3. Partitioning (Separation): This is the most critical step. The bound sequences (potential aptamers) must be efficiently separated from the unbound ones. Common methods: immobilizing the target on a column/beads, filtration through nitrocellulose filters (which trap protein-bound RNA/DNA), or capillary electrophoresis. 4. Elution (Recovery): The bound sequences are recovered, often by denaturing the complex (e.g., heating, changing pH, or using denaturants). 5. Amplification: The recovered sequences are amplified using PCR (for DNA aptamers) or Reverse Transcription-PCR (for…
1. Synthetic & Chemical Origin Fully In Vitro Production: Unlike biological antibodies produced in living cells (mice, rabbits, CHO cells), Adaptamers are synthesized chemically in a controlled laboratory setting. Precise Chemical Structure: Their composition, structure, and modifications are defined at the atomic level, leading to perfect batch-to-batch consistency. No Biological System Required: Production is independent of cell culture, fermentation, or animal use, making it more scalable and ethically straightforward. 2. Adaptive & Engineered Binding "Adapta-" Implies Engineering: The name suggests the molecule is engineered or evolved in vitro (like aptamers via SELEX) to bind with high affinity and specificity to a chosen target—proteins, small molecules, cells, etc. Tailor-Made: Their binding sites are not constrained by biological immune system mechanisms. They can be designed against targets that are poorly immunogenic, toxic, or highly conserved. 3. Core Molecular Scaffold (Likely Nucleotide or Peptide-Based) While unspecified, the most probable backbones are: Nucleic Acid-Based (like advanced aptamers): Composed of DNA or RNA, but heavily chemically modified (e.g., with 2'-fluoro, 2'-O-methyl, locked nucleic acids (LNA), or entirely synthetic bases) to confer nuclease resistance and enhanced stability. Peptide-Based (like peptidomimetics or constrained peptides): Composed of non-natural amino acids or cyclic structures for rigidity and protease resistance. Hybrid or Other Polymer: Could be a fusion of chemistries (e.g., oligonucleotide with a peptide-like side chain) for…
