Workflow for Aptamer Development Service

Aptamer Screening Service Workflow

This is a detailed explanation of the Aptamer Screening Service Workflow. Aptamer screening, also known as SELEX, is an iterative in vitro selection process used to isolate nucleic acid molecules with high affinity and specificity for a given target from a random oligonucleotide library.

A complete aptamer screening service typically follows this standardized workflow, which can be divided into several core stages:

Stage 1: Project Design and Initiation

1. Requirement Communication and Target Confirmation

   • Target Properties: Define target type (protein, small molecule, cell, virus, etc.), purity, concentration, and availability.

   • Screening Objective: Determine the intended application of the aptamers (diagnostics, therapeutics, detection, etc.), which dictates the stringency of the screening (e.g., the need for counter-selection/negative selection).

   • Chemical Modification: Decide whether to use modified nucleotides (e.g., 2′-F, 2′-O-Me) to enhance aptamer stability and nuclease resistance.

   • Library Design: Determine the length of the random region (typically 30-50 nt) and the flanking primer sequences.

2. Protocol Development and Contract Agreement

   • The service provider develops a detailed Experimental Protocol based on requirements, including the number of selection rounds, positive/negative selection strategy, separation methods, expected deliverables, etc.

   • Both parties finalize the protocol, timeline, cost, and sign a contract.

Stage 2: SELEX Selection Cycle (Core Process)

This is a repetitive cycle of “Binding-Separation-Amplification,” typically conducted for 8-15 rounds.

Basic Steps for Each Round:

1. Incubation and Binding

   • The initial single-stranded DNA or RNA library, containing a vast diversity of sequences (typically 10^14-10^15 variants), is incubated with the target under optimized binding conditions.

2. Separation

   • The target-aptamer complexes are separated from unbound sequences. This is a critical step. Common methods include:

     • Membrane Filtration: For large protein targets, complexes are retained on a filter membrane.

     • Magnetic Beads: The target is immobilized on magnetic beads, and complexes are separated using a magnetic field.

     • Affinity Chromatography: The target is pre-immobilized on a chromatography column.

     • Cell-SELEX: For cell-surface targets, unbound sequences are removed by centrifugation or washing.

3. Elution

   • High-affinity aptamers are eluted (released) from the target. Common methods include heating, denaturing agents, or high-salt buffers.

4. Amplification and Regeneration

   • PCR Amplification: The small amount of eluted aptamer sequences is amplified exponentially via PCR.

   • Single-Strand Preparation:

     • For DNA aptamers: Typically prepared for the next round via streptavidin-bead separation or asymmetric PCR.

     • For RNA aptamers: Requires in vitro transcription of DNA to RNA. Each round involves reverse transcription and transcription steps.

5. Monitoring and Evaluation

   • Monitor the increase in target-binding affinity of the enriched pool after each round (e.g., using an ELISA-like binding assay or flow cytometry).

   • The selection is terminated when the binding signal reaches a plateau or shows no significant further increase.

Stage 3: Cloning, Sequencing, and Analysis

1. Cloning

   • The enriched pool from the final selection round is cloned (typically into plasmids) and transformed into E. coli bacteria.

2. High-Throughput Sequencing

   • Perform Sanger sequencing on dozens to hundreds of individual clones, or subject the enriched pool directly to Next-Generation Sequencing to obtain massive sequence data.

3. Bioinformatics Analysis

   • Sequence Clustering: Group sequences based on homology to identify the most frequent families and representative sequences.

   • Motif Analysis: Predict common secondary structures (e.g., stem-loops, G-quadruplexes) crucial for binding.

   • Candidate Sequence Selection: Select several to dozens of representative sequences with high abundance and interesting structures for further validation.

Stage 4: Candidate Aptamer Validation and Characterization

1. Synthesis and Purification

   • Chemically synthesize the chosen candidate aptamers and purify them.

2. Affinity Measurement

   • Determine the dissociation constant (Kd) using methods like Surface Plasmon Resonance, Isothermal Titration Calorimetry, or Filter Binding Assay. Ideal Kd values are typically in the nanomolar (nM) to picomolar (pM) range.

3. Specificity Assessment

   • Test candidate aptamers for cross-reactivity against target analogs, unrelated proteins, or cell lines to ensure high specificity.

4. Functional Validation

   • Validate based on the intended application:

     • Detection/Diagnostics: Evaluate performance (sensitivity, limit of detection) in assays like ELISA, lateral flow, or biosensors.

     • Therapeutics/Inhibition: Test the ability to block protein-protein interactions or inhibit cellular functions.

Stage 5: Reporting and Delivery

1. Project Report

   • The service provider submits a detailed technical report containing:

     • Complete experimental procedures and parameters.

     • Monitoring data from the selection process.

     • Sequencing analysis results and the candidate sequence list.

     • Validation data (Kd values, specificity data, etc.).

     • Discussion and recommendations.

2. Material Delivery

   • Deliverables typically include:

     • The final enriched aptamer pool.

     • Sequence information for all validated candidate aptamers.

     • Synthesized and validated 1-3 core candidate aptamers as plasmid or lyophilized powder (as per contract agreement).

     • Relevant raw experimental data.

Service Workflow Summary Diagram

Client Needs → Protocol Design → Contract Signing
                                  ↓
                        SELEX Cycle (Rounds 1-n)
                        Bind → Separate → Elute → Amplify
                                  ↓
                   Monitor Enrichment → No → Continue Next Round
                                  ↓Yes
                        Cloning & HTS Sequencing
                                  ↓
                        Bioinformatics Analysis
                                  ↓
                  Candidate Synthesis & Validation
             (Affinity, Specificity, Function)
                                  ↓
                     Final Report & Material Delivery

Key Considerations When Choosing a Service Provider

• Experience and Case Studies: Successful experience with similar targets.

• Technology Platform: Separation methods, sequencing, and analytical capabilities.

• Chemical Modification Offering: Crucial for therapeutic applications.

• Depth of Validation: Whether comprehensive affinity, specificity, and functional validation are included.

• Project Timeline and Cost: A complete workflow typically takes 3-6 months. Costs vary based on target difficulty and depth of validation.

We hope this detailed workflow provides you with a comprehensive understanding of the aptamer screening service process.