Aptamer Screening Service-Multi-Round SELEX Screening

Core Concept: What is SELEX?

SELEX (Systematic Evolution of Ligands by EXponential Enrichment) is an iterative, in vitro selection process. It starts with a vast, random library of oligonucleotides (10^14 – 10^15 unique sequences) and, over multiple rounds, enriches for those that bind to the target.

Standard Multi-Round SELEX Screening Service Workflow

A full-service provider will typically manage the entire process, which can be broken down into key phases:

Phase 1: Project Design & Target Preparation

• Target Consultation: Defining the target (e.g., protein, small molecule, cell, virus). Critical discussion of target purity, immobilization strategy, and selection conditions (buffer, temperature, counter-selection).

• Library Design: Selection of a random library (e.g., 40-nt random core with fixed primer sites). Options include DNA, RNA (requiring reverse transcription), or modified libraries (e.g., with 2′-F pyrimidines for nuclease resistance).

• Immobilization Strategy: The service provider will choose the best method:

  • Immobilized Target: (Most common for proteins) Binding target to beads (streptavidin, Ni-NTA for His-tag) or columns.

  • Counter-Selection: Using negative control surfaces (e.g., blank beads, related but undesired proteins) to subtract non-specific binders.

Phase 2: The SELEX Cycle (Repeated 8-15 Rounds)

This is the core iterative screening process. Each round consists of:

1. Incubation: The oligonucleotide library is incubated with the target under defined conditions.

2. Partitioning: Separation of target-bound sequences from unbound ones (e.g., via washing beads, filtration, or capillary electrophoresis). This is the key selection pressure.

3. Elution: Recovery of bound sequences (e.g., by heat denaturation, protein denaturants, or competitive elution).

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

5. Purification: Generation of a single-stranded DNA library for the next round. (For RNA, this includes in vitro transcription).

Monitoring: The provider will monitor enrichment, typically using quantitative PCR (qPCR) to track the amount of recovered DNA/RNA increasing round-over-round, indicating successful selection.

Phase 3: Post-SELEX Analysis & Identification

• High-Throughput Sequencing (HTS/NGS): The pools from later rounds (e.g., round 8, 10, 12, 15) are sequenced using Next-Generation Sequencing.

• Bioinformatics Analysis: This is critical. The service includes:

  • Clustering & Alignment: Identifying families of related sequences.

  • Motif Discovery: Finding conserved sequence or structural motifs among enriched candidates.

  • Ranking Candidates: Selecting top 10-20 candidate aptamers based on frequency, fold-enrichment, and cluster dominance.

Phase 4: Validation & Characterization

• Synthesis & Screening: The top candidate aptamers are chemically synthesized and tested for binding.

• Primary Binding Assays: Using techniques like Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI), or Electrophoretic Mobility Shift Assay (EMSA) to determine dissociation constant (Kd), confirming nM-μM affinity.

• Specificity Testing: Against negative control targets or closely related molecules.

• Functional Assays (Optional/Dependent): Testing for antagonism/agonism, inhibition of protein function, or staining of target cells.

What to Look for in a Service Provider

1. Experience & Expertise: Proven track record with targets similar to yours (soluble proteins, membrane proteins, small molecules, whole cells).

2. Technology Platform: Ask about their partitioning method. Advanced methods can lead to better aptamers:

   • Capillary Electrophoresis (CE)-SELEX: Excellent for purity and stringency.

   • Cell-SELEX: Specialized for live cell targets.

   • Automated SELEX Platforms: Increases reproducibility and throughput.

3. End-to-End Service: Can they handle from design to validated candidates, including synthesis and initial in vitro testing?

4. Bioinformatics Capability: Robust analysis of NGS data is non-negotiable for modern SELEX.

5. Timeline & Cost: A typical multi-round SELEX project takes 3-6 months to deliver validated candidate aptamers. Costs can range from $20,000 to $60,000+, depending on target complexity, number of rounds, and depth of validation.

Deliverables You Should Expect

• A detailed report outlining the selection conditions and progress.

• NGS data and bioinformatics analysis report with candidate rankings.

• Sequences of the top 10-20 aptamer candidates.

• Validation data for 2-3 lead candidates (synthesized) including Kd values and specificity profiles.

• The physical synthesized aptamer samples (lyophilized).

Advantages of Using a Service vs. In-House

• Access to Expertise: Avoids the steep learning curve and protocol optimization.

• Advanced Platforms: Access to CE-SELEX, automation, and sophisticated NGS analysis.

• Time Savings: Frees up your lab’s resources for downstream application development.

• Higher Success Rate: Experienced providers know how to troubleshoot challenging targets (e.g., hydrophobic proteins, non-immunogenic targets).

Key Questions to Ask Potential Providers

• “What is your success rate with targets like mine?”

• “Can you share a case study or example data from a past project?”

• “What is your standard partitioning method and why?”

• “How many selection rounds are included, and what triggers the decision to stop?”

• “What specific binding assays do you use for Kd determination?”

• “What happens if enrichment is not achieved in the standard number of rounds?”

By partnering with a proficient Aptamer Screening Service, you leverage specialized technology and expertise to efficiently transform your target of interest into high-affinity, functional aptamer reagents for diagnostics, therapeutics, or basic research.