Aptamer Identification

The unique secondary and tertiary structures of aptamers provide the specificity to detect even small structural changes in the target molecule, including the presence or absence of methyl or hydroxyl groups or differences in enantiomeric configurations.

Aptamers that bind specific targets are identified through a process known as Systematic Evolution of Ligands by Exponential enrichment (SELEX) in which binding molecules are selected from a large and diverse library of nucleic acids (either DNAs or RNAs). In this process, the nucleic acid library is incubated with the target molecule. Non-binding nucleic acids are then washed away, leaving behind only the molecules that have a capacity to bind to the target molecule.

The nucleic acids that are not washed away are then used to create a new library of nucleic acids that is enriched for the subset that binds the desired target. Repeating this selection-cycle on each subsequent library with increasing stringency of binding (e.g. lower concentration of target), ensures that nucleic acids that bind to the target with both high specificity and high affinity are enriched.

Aptamers are short, single-stranded oligonucleotides (DNA or RNA) that bind to specific target molecules with high affinity and specificity. They are often called “chemical antibodies.” The process of discovering new aptamers is called aptamer identification or selection, and the primary method used is SELEX (Systematic Evolution of Ligands by EXponential enrichment).

Here’s a breakdown of the key steps, methods, and considerations:

1. The Core Process: SELEX

SELEX is an iterative, in vitro selection process that mimics natural evolution. The basic cycle involves:

1. Library Design: Start with a vast, random synthetic oligonucleotide library (typically 10¹³ to 10¹⁵ unique sequences). Each sequence is flanked by constant primer regions for amplification.

2. Incubation: The library is incubated with the target molecule (e.g., a protein, small molecule, cell).

3. Partitioning: Sequences that bind to the target are separated from unbound sequences. This is the most critical and challenging step.

4. Elution: Bound sequences are recovered (eluted) from the target.

5. Amplification: The eluted sequences are amplified by PCR (for DNA) or RT-PCR (for RNA) to create an enriched pool for the next round.

6. Repetition: Steps 2-5 are repeated for multiple rounds (usually 8-15), progressively enriching the pool for the tightest-binding sequences.

7. Cloning & Sequencing: The final enriched pool is cloned and sequenced to identify individual aptamer candidates.

8. Characterization: Candidate aptamers are synthesized and tested for binding affinity (Kd), specificity (against related targets), and function.

2. Key Variations of SELEX

To address different targets and challenges, many SELEX variants have been developed:

• Counter-SELEX: Includes negative selection steps against closely related molecules to improve specificity.

• Cell-SELEX: Uses whole living cells as targets to identify aptamers for cell-surface markers, without prior knowledge of the target protein. Great for cancer cell targeting.

• Capture-SELEX: For small molecule targets, the library is immobilized, and binding sequences are captured by the soluble target.

• Capillary Electrophoresis-SELEX (CE-SELEX): Uses capillary electrophoresis for highly efficient partitioning based on mobility shift, often yielding high-affinity aptamers in fewer rounds.

• High-Throughput Sequencing (HTS) SELEX: Uses deep sequencing throughout the rounds to monitor sequence evolution and identify winners without cloning.

• In Silico SELEX: Uses computational methods to model and predict aptamer-target interactions, often to prioritize candidates or guide library design.

3. Critical Considerations in Aptamer Identification

• Target Choice: The target must be pure, stable, and in its native conformation (especially for proteins). Immobilization (on beads, columns) can sometimes mask binding sites or cause non-specific binding.

• Stringency: Increasing washing rigor and reducing target concentration in later rounds selects for higher affinity binders.

• PCR Bias: Amplification can favor sequences that PCR efficiently, not just those that bind best. Careful optimization is needed.

• Truncation & Optimization: Selected full-length sequences often contain redundant primer regions. Truncation studies are done to find the minimal, functional core aptamer. Chemical modifications (e.g., 2′-F, 2′-O-Me in RNA) can also be introduced post-SELEX to enhance nuclease resistance.

4. Post-SELEX: Analysis & Characterization

• Bioinformatics: Analyze sequencing data from HTS-SELEX to find conserved motifs, families, and consensus structures.

• Affinity Measurements: Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), or filter binding assays are used to determine dissociation constant (Kd).

• Specificity & Cross-Reactivity: Test against structurally similar targets or other non-target molecules.

• Structural Analysis: Use techniques like NMR, X-ray crystallography, or in silico modeling to understand the binding mechanism.

5. Applications of Identified Aptamers

The identified aptamers have diverse applications:

• Diagnostics: Biosensors (aptasensors), point-of-care tests.

• Therapeutics: As drugs (e.g., Macugen for AMD), drug delivery vehicles.

• Research Tools: For protein detection, purification, or inhibition.

• Analytical Chemistry: As ligands in affinity chromatography.

6. Challenges & Future Directions

• Speed & Cost: Traditional SELEX can be laborious and take months. Efforts are focused on automation and microfluidics to accelerate the process.

• “Artificial” Nature: Aptamers identified in vitro may not function well in complex biological matrices (in vivo).

• Aptamers for Membrane Proteins: Difficult due to the hydrophobic nature of many membrane proteins.

• Cell-Specific Delivery: Current research focuses on identifying aptamers that facilitate targeted delivery to specific tissues or cells.

Conclusion

Aptamer identification via SELEX is a powerful platform technology for generating target-specific recognition elements. While the core principle remains constant, continuous innovations in selection methodologies, partitioning strategies, and data analysis are making the process faster, more efficient, and more successful, expanding the potential of aptamers in biotechnology and medicine.