Classical SELEX Service for Aptamer

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:

1. Incubation: A vast, random-sequence nucleic acid library (10^14 – 10^15 different sequences) is exposed to the target.

2. Partitioning: Unbound sequences are washed away; bound sequences are retained.

3. Elution: The bound sequences are recovered.

4. Amplification: The recovered sequences are amplified by PCR (for DNA) or RT-PCR (for RNA).

5. 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 conditions.

• Monitoring: Using techniques like qPCR or gel electrophoresis to monitor enrichment progress.

3. Post-SELEX Analysis & Delivery

• Cloning & Sequencing: After the final round, the enriched pool is cloned into bacteria, and individual clones are Sanger sequenced (typically 50-200 clones).

• Sequence Analysis: Identifying sequence families and consensus motifs.

• Candidate Selection: Delivering a list of 5-20 top candidate aptamer sequences.

• Primary Characterization: Often includes initial synthesis and testing of a few top candidates via a simple binding assay (e.g., Enzyme-Linked Oligonucleotide Assay – ELOA, or flow cytometry for cell targets) to confirm binding.

What the Client Provides & Receives

Client Input:

• The Target: Sufficient quantity and purity of the protein, small molecule, or cells.

• Project Goals: Desired affinity (Kd), specificity, and intended application (diagnostic sensor, therapeutic inhibitor, etc.).

• Funding: The service fee.

Service Output (Deliverables):

1. A detailed report of the SELEX process and conditions.

2. A list of enriched aptamer sequences with frequency analysis.

3. Data on the top 2-5 candidates: This usually includes basic binding affinity (Kd) measurement via a method like filter binding or fluorescence anisotropy, and sometimes specificity data against a related negative control.

4. The aptamer sequences themselves. Some packages include a small amount of the unmodified, chemically synthesized aptamer for initial validation.