The SELEX (Systematic Evolution of Ligands by EXponential enrichment) protocol is a method to identify DNA aptamers that specifically bind to a target molecule, such as proteins, small molecules, or even whole cells. Below is a general protocol for DNA aptamer SELEX:
Materials Required
Starting DNA library: A pool of single-stranded DNA (ssDNA) molecules with randomized sequences (typically 10^13 to 10^15 different sequences).
Target molecule: The molecule for which aptamers will be selected (e.g., proteins, small molecules, or cells).
Binding buffer: Typically a buffer containing salts (e.g., PBS or Tris buffer) with additives like magnesium, calcium, or other ions necessary for aptamer folding and binding.
Separation method: The method to isolate bound DNA from unbound DNA (e.g., filtration, centrifugation, or affinity column).
PCR reagents: For amplification of the selected DNA sequences.
Primers: Forward and reverse primers complementary to the flanking regions of the randomized library.
Polyacrylamide gel or agarose gel: For DNA recovery after PCR amplification.
General DNA Aptamer SELEX Protocol
- Initial DNA Library Preparation:
Library design: The initial library consists of ssDNA molecules (usually 20-80 random nucleotides) flanked by fixed sequences on both ends for PCR amplification.
Random region: The central region of the DNA molecules is randomized to ensure diversity in the library (e.g., 5’-AGCAGCACAGAGTGGATGXXX…XXXATGGACGAATATGGGT-3’, where XXX…XXX is the random region).
- Incubation with the Target Molecule:
Incubate the DNA library with the target molecule in the binding buffer. This step allows the DNA sequences that can form specific interactions with the target to bind.
The incubation time is typically between 30 minutes to 1 hour, depending on the target, at room temperature or 37°C.
- Separation of Bound and Unbound DNA:
After incubation, separate the bound sequences (DNA aptamers that have interacted with the target) from unbound DNA.
Separation methods:
For protein or small molecule targets: This could involve filtration, affinity columns, magnetic beads, or chromatography.
For cell SELEX: Wash cells to remove unbound sequences and recover cells with bound DNA sequences.
Increase stringency over the course of the experiment by adjusting wash conditions (e.g., higher salt concentration or more washing steps) to remove weaker binders.
- Elution of Bound DNA:
Once bound and unbound sequences are separated, the bound DNA needs to be eluted. This can be done by heating (if heat-stable), changing the pH, or adding a competing ligand or salt solution to disrupt the aptamer-target interaction.
- Amplification of Eluted DNA:
The eluted DNA is then amplified by PCR to enrich for sequences that successfully bound to the target.
Use specific primers complementary to the flanking regions of the library.
Optimize PCR conditions to avoid amplification of non-specific products and ensure correct amplification of the aptamer pool.
Verify the PCR product on an agarose or polyacrylamide gel.
- Conversion to Single-Stranded DNA:
After PCR amplification, the product is double-stranded DNA (dsDNA). This needs to be converted back to single-stranded DNA (ssDNA) for the next round of SELEX.
Methods to generate ssDNA:
Asymmetric PCR: Use an excess of one primer to generate ssDNA.
Magnetic beads: If one primer is biotinylated, the dsDNA can be separated using streptavidin-coated beads, and the non-biotinylated strand can be eluted.
Denaturing PAGE gel: Separate the strands based on size difference.
- Repetition of SELEX Rounds:
Repeat steps 2 through 6 for several rounds (typically 8–15 rounds). With each round, the stringency is increased to select for DNA sequences with higher affinity and specificity.
Increase stringency by reducing the concentration of target molecules, increasing the number or strength of washes, or shortening the incubation time.
- Monitoring the Enrichment Process:
After several rounds of SELEX, monitor the binding affinity of the enriched DNA pool to the target using methods like:
Electrophoretic Mobility Shift Assay (EMSA): To observe DNA-target binding.
Surface Plasmon Resonance (SPR): To measure binding kinetics.
Fluorescence-based assays: To measure aptamer binding if the target is fluorescently labeled.
- Cloning and Sequencing of Final Pool:
After the final round of selection, clone the enriched pool of aptamers into a suitable vector and sequence individual clones.
Sequence analysis is performed to identify individual aptamers with unique sequences.
Multiple aptamer sequences are obtained and grouped into families based on sequence similarity.
- Characterization of Selected Aptamers:
Test individual aptamers for their affinity and specificity toward the target.
Dissociation constant (Kd): Measure using binding assays like fluorescence or SPR to determine the strength of aptamer-target interaction.
Assess specificity by testing aptamers against similar targets or control molecules to ensure the aptamer binds specifically to the target of interest.
Optional: Negative Selection (Counter-SELEX)
In some cases, to improve specificity, negative selection is introduced by incubating the DNA library with non-target molecules (or cells) to remove sequences that bind non-specifically.
The unbound sequences from the negative selection are then used for the next round of positive selection with the target.
Notes:
Target diversity: SELEX can be performed against various targets, including proteins, small molecules, ions, and whole cells.
Aptamer modifications: Post-selection, aptamers may be chemically modified to improve their stability, such as adding functional groups or using modified nucleotides.
This general DNA aptamer SELEX protocol can be adapted based on the specific target and application