Aptamer screening service:Technical process of Aptamer synthesis

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.

1. 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 ~ 1.2×10²⁴ possible sequences).

   • Constant Regions: Essential for PCR amplification during later steps.

2. 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:

1. Incubation: The synthetic library is incubated with the target molecule (protein, small molecule, cell, etc.).

2. Partitioning: The critical step. Methods to separate bound from unbound sequences include:

   • Immobilization: Target is fixed on beads, columns, or plates. Unbound sequences are washed away.

   • Filter Binding: Aptamer-target complexes are retained on nitrocellulose filters.

   • Capture Methods: Using biotin-tagged targets pulled down by streptavidin.

   • CE-SELEX: Using capillary electrophoresis to separate based on mobility shift.

3. Elution: Bound sequences are recovered, often by denaturing (heat, high salt, chaotropic agents) or specific competitive elution.

4. Amplification: The eluted pool is amplified:

   • For DNA: Direct PCR.

   • For RNA: Reverse transcription to DNA, then PCR, followed by in vitro transcription back to RNA.

5. Purification: The amplified product is purified (e.g., gel electrophoresis, column purification) to obtain the correct single-stranded form for the next round.

6. Counter-Selection (Negative Selection): Often included to increase specificity. The pool is incubated with non-target molecules or similar structures to remove cross-reactive binders before the target incubation.

Iteration: Steps 1-5 are repeated (typically 5-15 rounds). Stringency (washing harshness, target concentration) is increased over rounds to select for the tightest binders.

Phase 3: Post-SELEX Analysis, Optimization, and Production

1. Cloning & Sequencing: The final enriched pool is cloned into bacteria or sequenced via High-Throughput Sequencing (HTS). HTS reveals the consensus families of sequences that “won” the selection.

2. Characterization: Individual candidate sequences are chemically synthesized and tested for:

   • Affinity: Measured by Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), or filter binding assays (Kd values).

   • Specificity: Testing against related but non-target molecules.

   • Function: Does it inhibit an enzyme, block a receptor, etc.?

3. Truncation & Minimization: The full selected aptamer (with primers) is analyzed to find the shortest functional domain. Unnecessary nucleotides are removed to reduce cost and improve stability/yield.

4. Chemical Modification & Optimization:

   • For Stability (Therapeutic/Diagnostic Use): Nucleotides are modified post-selection to resist nucleases (e.g., 2′-Fluoro, 2′-O-Methyl ribose substitutions, inverted dT caps, PEGylation). Note: Some modern methods like SELEX with Modified Libraries (e.g., SOMAmers) incorporate modified nucleotides during the selection process.

   • Labeling: Attachment of fluorophores, biotin, or other reporter molecules for detection applications.

5. Scale-Up Production: The finalized sequence is mass-produced via large-scale solid-phase chemical synthesis and purified by HPLC or PAGE, ensuring lot-to-lot consistency.

Modern Variations & Key Technologies

• Automated SELEX: Robotic platforms to handle many rounds and targets in parallel.

• Cell-SELEX: Uses whole living cells as targets to find aptamers for specific cell states (e.g., cancer cells).

• High-Throughput Sequencing (HTS): Revolutized analysis, allowing deep mining of the selection landscape and identification of rare, high-quality aptamers.

• In Silico Design & Analysis: Bioinformatics tools (e.g., AptaGUI, FASTAptamer) to analyze HTS data, predict secondary structures (folding), and model 3D interactions.

Comparison: DNA vs. RNA Aptamer Synthesis

Summary of the Technical Workflow:

Design Library → Chemical Synthesis → Iterative SELEX (Binding/Partitioning/Amplification) → HTS & Cloning → Characterization → Truncation/Optimization → Chemical Modification → Scale-Up Synthesis.