Aptamer Screening Service- Capture-SELEX

1. Core Concept: What is Capture-SELEX?

Capture-SELEX (Systematic Evolution of Ligands by EXponential Enrichment) is an advanced selection technique used to discover single-stranded DNA or RNA aptamers that bind to a specific target molecule.

The key innovation is that the target molecule is immobilized (or “captured”) on a solid support via a short, known oligonucleotide sequence that is part of the initial random library. This makes it exceptionally powerful for selecting aptamers against small molecules or targets without natural immobilization sites.

2. The Key Differentiator: How It Differs from Classical SELEX

• Classical SELEX: The target itself is immobilized directly on a surface (e.g., a bead or plate). This can sometimes lead to aptamers that bind to the surface or the immobilized region of the target, which may not function well in solution.

• Capture-SELEX: The library itself is immobilized via a complementary “capture sequence.” Only sequences that bind to the free, unmodified target in solution undergo a conformational change that releases them from the capture strand for collection.

3. Step-by-Step Process of a Capture-SELEX Service

A service provider will typically manage this entire pipeline:

Step 1: Project Design & Library Synthesis

• You define the target (e.g., a small molecule, protein, cell).

• The service designs a custom single-stranded DNA (ssDNA) library: [5' Fixed Primer Sequence - RANDOM Region (40-60 nt) - 3' Fixed Primer Sequence - CAPTURE Sequence].

• The Capture Sequence is a short, known sequence designed to bind reversibly to a complementary oligonucleotide immobilized on beads.

Step 2: Immobilization of the Library

• The ssDNA library is hybridized to its complementary “capture oligonucleotides” that are chemically attached to magnetic beads or a column.

• The library is now tethered to a solid support.

Step 3: Selection Rounds (The Cyclic Core)

1. Negative Selection (Optional but common): Incubate the immobilized library with any relevant negative matrices (e.g., blank beads, similar molecules) to remove sequences that bind non-specifically.

2. Positive Selection: Incubate the immobilized library with the free, soluble target.

   • Key Event: Aptamer candidates within the random pool that bind to the target undergo a conformational change. This change often weakens or disrupts the hybridization with the immobilized capture strand.

3. Elution: The strongest binders, now released from the beads into the solution, are collected. This is the critical step—binding to the target directly triggers elution.

4. Amplification: The eluted sequences are PCR-amplified.

5. Regeneration: The double-stranded PCR product is converted back into a single-stranded library, ready for the next round of selection (re-hybridized to fresh capture beads).

Step 4: Monitoring & Stringency Increase

• The service monitors enrichment (e.g., via qPCR or sequencing) over 8-15 rounds.

• Selection stringency is increased by reducing target concentration, increasing wash times, or adding counter-selection steps to drive the evolution of high-affinity, specific aptamers.

Step 5: Sequencing & Bioinformatics

• High-Throughput Sequencing (NGS) is performed on the enriched pools.

• Bioinformatic analysis identifies candidate aptamer families based on sequence homology, frequency, and predicted secondary structures.

Step 6: Candidate Validation

• The service synthesizes the top 10-20 candidate aptamers.

• They perform binding assays (e.g., ELISA-style, SPR, BLI) to confirm affinity (Kd) and specificity against the target and related molecules.

• The final deliverable is a shortlist of validated aptamer sequences with characterized performance data.

4. Advantages of Using a Capture-SELEX Service

• Ideal for Small Molecules: Excellent for targets <1000 Da that are hard to immobilize directly.

• Solution-Phase Binding: Selects for aptamers that recognize the target in its native, soluble state.

• Reduced Surface Bias: Minimizes selection of aptamers that bind to the immobilization matrix.

• Conformational Selection: Inherently selects for binding-induced structural changes, which is useful for biosensor applications.

• Expertise & Infrastructure: Access to specialized NGS, robotics, and bioinformatics without capital investment.

• Faster Timeline: Typically 3-6 months from start to validated candidates.

5. Typical Service Deliverables

A full-service package usually includes:

• Project consultation and design.

• Custom library synthesis.

• Complete Capture-SELEX procedure (8-15 rounds).

• Next-Generation Sequencing and bioinformatic analysis.

• Synthesis and preliminary validation (Kd, specificity) of top aptamer candidates.

• A final report with all sequences, data, and protocols.

6. Applications of Aptamers Discovered via Capture-SELEX

• Diagnostic Sensors: (e.g., for environmental toxins, drugs, metabolites).

• Therapeutic Agents: Antagonists or agonists for disease targets.

• Drug Delivery: Targeted delivery vehicles.

• Analytical Reagents: Replace antibodies in assays (ELISA, Flow Cytometry).

In summary, a Capture-SELEX service provides a streamlined, expert-driven path to generate high-quality aptamers, particularly for challenging targets like small molecules, by leveraging a clever selection mechanism that ensures the identification of binders functional in solution.