What is Classical SELEX? SELEX is an iterative, in vitro selection process used to isolate single-stranded DNA or RNA molecules (aptamers) that bind with high affinity and specificity to a target (e.g., a protein, small molecule, cell, or virus). The "classical" method refers to the original, well-established protocol involving: Incubation: A vast, random-sequence nucleic acid library (10^14 - 10^15 different sequences) is exposed to the target. Partitioning: Unbound sequences are washed away; bound sequences are retained. Elution: The bound sequences are recovered. Amplification: The recovered sequences are amplified by PCR (for DNA) or RT-PCR (for RNA). Repetition: This cycle (typically 8-15 rounds) is repeated, enriching the pool for the strongest binders. Components of a Classical SELEX Service A full-service provider typically manages the entire pipeline: 1. Project Design & Consultation Target Characterization: Discussing the target's properties (purity, stability, availability). Selection Strategy: Deciding on immobilization method (e.g., target immobilized on beads, or "counter-SELEX" to eliminate binders to the immobilization matrix or similar non-target molecules). Library Design: Choosing DNA or RNA, length of the random region (typically 20-60 nt), and fixed primer regions. 2. The SELEX Process Execution Library Synthesis: Chemical synthesis of the initial random library. Cycle Management: Performing the repetitive rounds of binding, washing, elution, and amplification under optimized buffer and stringency…
What is an Aptamer? An aptamer is a short, single-stranded oligonucleotide (DNA or RNA) or peptide that binds to a specific target molecule (e.g., proteins, small molecules, cells, viruses) with high affinity and specificity. Often called "chemical antibodies," they offer advantages like stability, low-cost synthesis, and minimal batch-to-batch variation. The Core Process: SELEX The standard method for aptamer selection is SELEX (Systematic Evolution of Ligands by EXponential enrichment). Basic SELEX Workflow: Library Synthesis: Create a vast random-sequence oligonucleotide library (typically 10¹³ - 10¹⁵ unique sequences) flanked by constant primer regions for PCR amplification. Incubation: The library is incubated with the target molecule under controlled conditions (buffer, temperature, time). Partitioning: Bound sequences are separated from unbound ones. This is the most critical step and varies based on target (e.g., filtration, affinity columns, magnetic bead separation). Elution: Bound aptamers are recovered from the target (e.g., by denaturation or competitive elution). Amplification: The recovered pool is amplified by PCR (for DNA) or RT-PCR (for RNA) to create an enriched library for the next round. Iteration: Steps 2-5 are repeated (typically 8-15 rounds) to progressively enrich for sequences with the highest affinity and specificity. Cloning & Sequencing: The final enriched pool is cloned and sequenced to identify individual aptamer candidates. Key Variants of…
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…
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…
Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide…
KMD Bioscience offers comprehensive aptamer discovery services that cover the full workflow—from nucleic acid aptamer design and synthesis, to multi-target SELEX screening, to final affinity and specificity validation. With advanced laboratory platforms and experienced scientists, we provide reliable and efficient one-stop aptamer solutions for diagnostics, therapeutics, biosensors and research applications. Nucleic Acid Aptamer Introduction Aptamers are single-stranded nucleic acid molecules (DNA or RNA) that fold into stable three-dimensional structures capable of binding specific targets with high affinity. Compared with antibodies, aptamers offer several advantages, including small molecular size, low immunogenicity, chemical synthesis, batch consistency and flexible modification. They can bind diverse targets such as proteins, peptides, cells, small molecules and ions, making them valuable tools in biotechnology and analytical applications. DNA Aptamer: DNA aptamers are aptamers composed of deoxyribonucleic acid (DNA). They usually have a double helix structure and are composed of four bases (adenine A, thymine T, guanine G, cytosine C) connected by phosphodiester bonds. They are single-stranded oligonucleotide sequences, typically with 56-120 bases, that bind the target sequence efficiently by recognizing specific spatial structures. DNA aptamers are widely used in biological analysis, biomedicine, and other fields because of their stability and easy chemical modification. RNA Aptamers: RNA aptamers are aptamers…
KMD Bioscience can provide a range of services for aptamer development, SELEX screening and optimization of aptamers, which are mainly based on aptamer SELEX screening technology. The target molecules of aptamer range widely, including small molecules such as metal ions, amino acids and nucleotides, biological macromolecules such as growth factors, proteins and peptides, and even viruses, bacteria, living cells, tissue sections and living organisms. KMD Bioscience can provide suitable aptamer SELEX screening services for a variety of targets, including peptides, and continue to advance in aptamer development. Peptides are polymers formed by covalent linkage of two or more amino acids through peptide bonds. According to the number of amino acid residues, peptides can be divided into oligopeptides (generally composed of 2 to 10 amino acids) and peptides (composed of more than 10 amino acids). Peptides have a variety of functions in organisms, such as hormones, neurotransmitters, growth factors, etc. They play an important role in regulating the physiological activities of organisms and participating in metabolic processes. Nucleic acid aptamers obtained by screening peptides have a variety of uses. For example, based on its recognition function, it can be used as an affinity reagent to establish analytical detection methods or carry out biological imaging studies. Based…