Aptamer Screening Service-NGS-SELEX

Core Concept of NGS-SELEX

Traditional SELEX uses a few rounds of selection and cloning/Sanger sequencing of a handful of clones. NGS-SELEX performs deep sequencing (millions to billions of reads) at every selection round. This allows you to:

1. Track the entire evolution of the oligonucleotide pool in real-time.

2. Identify enriched sequences and families early.

3. Perform sophisticated bioinformatics analysis to find winners, not just rely on final round abundance.

4. Dramatically reduce the number of selection rounds needed (often 3-6 rounds instead of 8-15).

Standard Service Workflow

A full-service provider would typically offer the following pipeline:

1. Project Design & Library Synthesis

• Consultation: Target properties (protein, small molecule, cell), desired aptamer properties (Kd, specificity, buffer conditions).

• Library Design: Standard (40-60 nt random region) or custom (doped libraries, modified nucleotides like 2′-F, 2′-OMe, SOMAmers).

• Primer & Library Synthesis: Providing the initial, highly diverse DNA or RNA library (10^14 – 10^15 unique sequences).

2. SELEX Selection

• Immobilization: Immobilizing the target (on beads, column, plate) or using solution-based techniques (capture-SELEX, toggle-SELEX).

• Counter-Selection: Including steps to remove binders to immobilization matrix or off-targets.

• Stringency Control: Increasing selection pressure over rounds (e.g., reduced target concentration, increased wash stringency).

• Amplification: Careful PCR (with optimization to minimize bias) to regenerate the pool for the next round.

3. NGS & Core Bioinformatics

• Sample Preparation: Preparing sequencing libraries from the selected pools of each round (and sometimes the negative selection eluates).

• High-Throughput Sequencing: Using platforms like Illumina MiSeq/HiSeq to generate millions of reads per sample.

• Primary Analysis:

  • Demultiplexing & quality control.

  • Clustering reads into unique sequences.

  • Tracking the frequency and enrichment of each unique sequence across rounds. This generates massive fold-enrichment plots.

4. Advanced Bioinformatics & Aptamer Identification

This is where the true power of NGS shines. Service providers use algorithms to:

• Cluster sequences into families based on shared motifs and secondary structure.

• Identify conserved consensus sequences and structural motifs.

• Rank candidate aptamers not just by final abundance, but by enrichment kinetics (e.g., sequences that appear early and rise consistently are often high-affinity binders).

• Generate a final list (e.g., 50-200) of candidate sequences for synthesis and validation.

5. Validation (Often as an Add-On Service)

• Candidate Synthesis: Synthesis of 20-100 top candidate aptamers (usually DNA, or modified RNA).

• Primary Screening: Using a rapid, medium-throughput method like ELONA (Enzyme-Linked Oligonucleotide Assay) or BLI/SPR in a multiplexed format to confirm binding.

• Characterization: For confirmed binders, determining:

  • Affinity (Kd) via titration using BLI, SPR, or flow cytometry (for cells).

  • Specificity against related targets or control cells.

  • Optimization: Truncation to find the minimal functional sequence.

Key Advantages of Using an NGS-SELEX Service

• Higher Success Rate: De-risks aptamer discovery, especially for difficult targets.

• Speed: Fewer selection rounds and intelligent candidate selection shorten project timelines.

• Rich Data: Provides a complete evolutionary map of the selection, invaluable for troubleshooting and publications.

• Discovery of Rare but High-Quality Aptamers: Finds excellent binders that might not be the most abundant in the final pool.

What to Look for in a Service Provider

1. Experience with Your Target Type: (Proteins, small molecules, whole cells, viruses). Cell-SELEX is particularly specialized.

2. Bioinformatics Capability: This is critical. Ask about their analysis pipeline and how they prioritize candidates.

3. Modification Expertise: If you need nuclease-resistant aptamers (for therapeutics/diagnostics), ensure they can work with modified nucleotide libraries (e.g., 2′-F, 2′-OMe pyrimidines).

4. Validation Options: A service that seamlessly integrates selection, sequencing, and validation is ideal.

5. Data Deliverables: Clarify what you get—raw sequencing data, analysis reports, candidate lists, alignment files, etc.

Potential Challenges & Considerations

• Cost: More expensive than traditional SELEX due to sequencing and bioinformatics.

• Bioinformatics Complexity: The volume of data can be overwhelming without expertise.

• Amplification Bias: PCR bias during SELEX can still skew results; a good service will optimize this.

• False Positives from Bioinformatics: In silico candidates must be validated experimentally.

Leading Companies & Labs Offering Services (Examples)

• Aptamer Sciences

• Aptagen

• Base Pair Biotechnologies

• TriLink BioTechnologies (with modified nucleotides)

• AM Biotech

• Many academic core facilities and specialized labs also offer contract SELEX.

In summary, an NGS-SELEX service is a powerful, turn-key solution for generating high-quality aptamers with a data-driven approach. It is the recommended path for any serious aptamer development project for diagnostics, therapeutics, or research tools.