Aptamer Screening: Current Methods and Future Trend towards Non-SELEX Approach

Aptamers are short, single-stranded oligonucleotides (DNA or RNA) that bind to specific targets (proteins, small molecules, cells) with high affinity and specificity. Their generation has traditionally been dominated by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. However, this method has significant limitations, driving innovation toward non-SELEX approaches.

1. Current Predominant Method: SELEX

SELEX is an iterative, in vitro selection-amplification process with several variations, each suited for different targets and applications.

Core Steps of SELEX:

1. Library Creation: A random oligonucleotide library (10¹³–10¹⁵ sequences) flanked by fixed primer sites.

2. Incubation & Binding: Library exposed to the target.

3. Partitioning: Separation of target-bound sequences from unbound ones.

4. Amplification: PCR (DNA) or RT-PCR (RNA) of bound sequences.

5. Repetition: Typically 8–15 cycles to enrich high-affinity binders.

6. Cloning & Sequencing: Identification of enriched aptamer candidates.

Key Variations of SELEX:

• Cell-SELEX: Uses whole cells as targets, ideal for biomarker discovery.

• Capture-SELEX: For small molecules, using immobilized targets.

• Capillary Electrophoresis-SELEX (CE-SELEX): High-efficiency partitioning via electrophoresis.

• Microfluidic-SELEX: Reduces time and reagent use via automation.

• Magnetic Bead-Based SELEX: Easy separation using target-coated beads.

Limitations of SELEX:

• Time-Consuming: Multiple cycles take weeks to months.

• Labor-Intensive: Requires significant hands-on effort.

• Amplification Bias: PCR can favor certain sequences unrelated to binding.

• Limited Sequence Diversity: Early high-affinity binders can dominate, reducing diversity.

• Cost: High reagent and time costs.

2. Emerging Trend: Non-SELEX Approaches

Non-SELEX methods aim to bypass iterative selection-amplification cycles, reducing time, cost, and bias. These are largely single-round selection techniques coupled with high-throughput sequencing (HTS) and bioinformatics.

A. Key Non-SELEX Strategies:

1. High-Throughput Sequencing & Bioinformatics Mining:

   • Method: A single round of selection followed by deep sequencing (NGS) of bound sequences.

   • Analysis: Bioinformatics tools identify enriched families, predict structures, and simulate binding.

   • Advantage: Captures a broader sequence space; avoids PCR bias across cycles.

2. Aptamer Discovery via Structure-Switch Mechanism:

   • Method: Libraries designed to change conformation upon target binding, directly signaling selection without partitioning.

   • Example: Structure-Switching SELEX (SS-SELEX) reduces rounds by using a conformational shift for easy separation.

3. Magnetic Activation of Cell Sorting (MACS)-Based Single-Round Selection:

   • Method: Target cells labeled with a library, and bound sequences are isolated via magnetic sorting in a single stringent round.

   • Advantage: Fast, suitable for complex cellular targets.

4. Microfluidic/Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM):

   • Method: Uses capillary electrophoresis to separate bound/unbound complexes in a single step based on mobility shifts.

   • Advantage: Extremely efficient partitioning; often requires only 1–3 rounds.

5. In Silico Selection & Rational Design:

   • Method: Computational modeling of target structure to design aptamers de novo or refine selected candidates.

   • Tools: Molecular docking, molecular dynamics, machine learning algorithms.

   • Future Potential: Integration with AI to predict binding sequences from target structural data.

B. Advantages of Non-SELEX Approaches:

• Speed: Selection in days vs. weeks.

• Reduced Bias: Minimizes PCR and sampling artifacts.

• Cost-Effectiveness: Less reagent use, fewer rounds.

• Rich Data: HTS provides massive datasets for better candidate identification and understanding of binding motifs.

3. Future Trends & Integration

The future lies in hybrid and convergent strategies that leverage the strengths of both SELEX and non-SELEX methodologies.

1. SELEX-lite (Minimal Round SELEX):

   • Combine 1–2 rounds of physical selection with HTS and computational enrichment.

2. Machine Learning & AI-Driven Discovery:

   • Train models on existing aptamer-target databases to predict binding sequences.

   • Generative AI could design novel aptamer libraries optimized for specific targets.

3. Single-Molecule & Real-Time Sequencing Integration:

   • Use platforms like Oxford Nanopore to monitor binding events in real time without amplification.

4. Cell-Free Display Technologies:

   • Ribosome display or mRNA display for proteins adapted for nucleic acids, allowing continuous evolution without PCR.

5. Functional Screening via Synthetic Biology:

   • Couple aptamer libraries to gene circuits in cells, where binding triggers a selectable phenotype.

6. Point-of-Care Aptamer Generation:

   • Portable microfluidic devices for on-demand aptamer selection in clinical or field settings.

4. Challenges in Non-SELEX Development

• Need for Robust Bioinformatics: Dependent on algorithms for candidate identification.

• Validation: High-throughput methods require careful experimental validation of binding and function.

• Limited Complexity Handling: For extremely complex targets (e.g., whole tissues), iterative enrichment may still be necessary.

• Standardization: Lack of standardized protocols compared to SELEX.

Conclusion

The field of aptamer screening is transitioning from iterative, labor-intensive SELEX to rapid, data-driven non-SELEX approaches. The integration of high-throughput sequencing, microfluidics, and artificial intelligence is enabling a new generation of aptamer discovery that is faster, cheaper, and more comprehensive. While traditional SELEX remains a reliable workhorse, especially for novel targets with unknown receptors, the future is clearly leaning towards streamlined, single-round selections augmented by computational power. The ultimate goal is a fully automated, AI-integrated platform that can deliver high-affinity aptamers for any target within days.