How to Obtain Aptamers? — SELEX Technology

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 RNA aptamers).

• This increases the quantity of the selected sequences for the next round.

6. Conditioning & Stringency:

• With each round, the selection conditions become more stringent (e.g., reduced incubation time, increased wash strength, introduction of competitive molecules) to selectively favor the sequences with the highest affinity and specificity.

• Counter-Selection: Often included, where the pool is incubated with related but undesired targets (e.g., a similar protein or the immobilization matrix alone) to remove cross-reactive binders. Only the unbound fraction proceeds to bind the real target.

7. Cloning, Sequencing, and Characterization:

• After the final round, the enriched pool is cloned and individual sequences are determined.

• These candidate aptamers are synthesized individually and their binding affinity (Kd), specificity, and function are rigorously tested in vitro.

Key Variations of SELEX (To Overcome Challenges)

Classic SELEX has limitations (length, time, bias). These advanced methods address them:

• Capillary Electrophoresis-SELEX (CE-SELEX): Uses capillary electrophoresis for ultra-efficient partitioning based on mobility shift. Very fast (2-4 rounds) and high affinity.

• Cell-SELEX: Uses whole living cells as targets. Crucial for generating aptamers that recognize native cell-surface proteins in their natural conformation. Vital for cancer cell targeting.

• Toggle-SELEX: Alternates selection between two related targets (e.g., human and mouse protein) to generate aptamers that recognize a conserved epitope, improving cross-reactivity.

• Automated & High-Throughput SELEX: Uses robotics to handle multiple selections in parallel, drastically increasing speed and reproducibility.

• In Silico SELEX & Next-Generation Sequencing (NGS):

  • NGS is now used during SELEX rounds to track pool evolution, identify enriched families early, and prevent over-enrichment of a few sequences.

  • Bioinformatics tools analyze NGS data to predict aptamer structures and identify consensus binding motifs.

• Genetically Encoded Libraries: For very complex targets, libraries can be expressed on the surface of phages or yeast, linking the aptamer sequence to its binding function.

The Post-SELEX Process: Optimization

Once a lead aptamer sequence is identified, it is often modified for practical use:

• Truncation: Finding the minimal essential binding sequence.

• Stabilization:

  • For RNA aptamers: Using 2′-fluoro or 2′-O-methyl modified nucleotides to resist RNase degradation.

  • For DNA aptamers: Capping ends or using modified backbones.

• Linking: Conjugating to reporters (fluorophores), drugs, or nanoparticles for applications.

Summary: The Aptamer Discovery Pipeline

1. Define Target & Conditions. (What are you binding to? Under what buffer/physiological conditions?)

2. Choose SELEX Strategy. (Standard, Cell-based, CE-SELEX, etc.)

3. Perform Iterative SELEX Rounds. (Incubate → Partition → Elute → Amplify, with increasing stringency).

4. Analyze Enriched Pool. (Clone & Sanger sequence, or use NGS for deep analysis).

5. Identify Candidate Sequences. (Look for consensus families from sequencing data).

6. Characterize & Optimize. (Synthesize individual aptamers, measure Kd, test specificity, truncate, stabilize).

7. Validate in Application. (Test in sensing, therapeutic, or imaging assays).

In essence, SELEX is a powerful combinatorial chemistry platform that mimics natural evolution, enabling the discovery of nucleic acid-based binding molecules (aptamers) against an astonishingly wide range of targets, from small molecules to whole cells. Its continuous evolution with new technologies makes it faster, more efficient, and more powerful than ever.