The SELEX (Systematic Evolution of Ligands by Exponential enrichment) protocol is a method used to identify aptamers—nucleic acid molecules (DNA, RNA, or modified versions) that can bind specifically to a target molecule. In the context of cell SELEX, the protocol is adapted to select aptamers that can bind specifically to cell surface markers, which is particularly useful for distinguishing different cell types, including cancer cells. Below is a general outline of a cell SELEX protocol:
Materials Required
Cell cultures: Target cells and control cells (if used for negative selection)
Starting oligonucleotide library: A randomized DNA or RNA library, typically containing 10^13–10^15 different sequences.
Binding buffer: A buffer that supports cell viability while promoting binding interactions (often PBS or a similar buffer supplemented with proteins like BSA to prevent non-specific binding)
PCR reagents: For amplification of selected sequences
Primers: Forward and reverse primers specific to the starting oligonucleotide sequences
Fluorescent dyes: For flow cytometry analysis
Column or magnetic beads: For cell separation
General Cell SELEX Protocol
- Initial Oligonucleotide Library Preparation:
Begin with a highly diverse library of oligonucleotides (randomized sequences flanked by known primer regions for PCR amplification). This serves as the pool from which specific aptamers will be selected.
- Incubation with Target Cells:
Incubate the randomized oligonucleotide library with the target cells (live or fixed) under conditions that maintain cell viability and allow binding interactions.
Incubation time typically ranges from 30 minutes to 1 hour at room temperature or 37°C.
- Washing to Remove Unbound Sequences:
After incubation, wash the cells multiple times to remove unbound or weakly bound oligonucleotides. The number and stringency of washes increase in subsequent rounds of SELEX to enhance the specificity of selected aptamers.
- Elution of Bound Sequences:
Elute the oligonucleotides that remain bound to the target cells. This can be done by heat denaturation, increasing salt concentration, or using a competing molecule.
- Amplification of Bound Oligonucleotides:
Use PCR (for DNA aptamers) or RT-PCR (for RNA aptamers) to amplify the pool of sequences that bound to the target cells. This enriched pool will be used for subsequent rounds of selection.
Monitor amplification by gel electrophoresis or quantitative PCR to ensure correct amplification.
- Counter Selection (Optional):
To improve the specificity of the selected aptamers, a negative selection step (counter-SELEX) can be included. This involves incubating the library with control cells (e.g., healthy cells or non-target cells). Sequences that bind to control cells are discarded, and the unbound sequences are used for further rounds of selection.
- Repetition of Selection Cycles:
Repeat steps 2 to 6 for several cycles (typically 6–12 cycles). In each cycle, the stringency of the selection process is increased (e.g., more washes, decreased incubation time), ensuring that only the best-binding sequences are retained.
- Monitoring Selection Progress:
After each round of SELEX, the binding affinity and specificity of the enriched pool can be assessed using techniques like flow cytometry, confocal microscopy, or surface plasmon resonance (SPR) to determine the success of the selection.
- Cloning and Sequencing of Final Pool:
After the final round of selection, the enriched oligonucleotide pool is cloned into a suitable vector and sequenced to identify individual aptamer sequences.
- Binding Assay of Selected Aptamers:
The binding affinity and specificity of individual aptamers are tested against the target cells using methods like flow cytometry, confocal microscopy, or other biophysical techniques. The dissociation constant (Kd) is often measured to evaluate the binding strength.
Notes
Positive vs Negative Selection: It is important to design the SELEX protocol to avoid selecting aptamers that bind to common structures on cells unrelated to the desired target. This is achieved through counter-selection steps where aptamers that bind to non-target cells (e.g., healthy cells or irrelevant cells) are eliminated.
Optimization: Stringency of washing and the number of SELEX rounds are critical factors and need to be optimized depending on the target cell type and the desired specificity of the aptamers.
This general protocol can be adapted to different target cells, and variations may be implemented based on the type of aptamers being selected (DNA vs RNA) or the specific requirements of the experiment (e.g., in vivo SELEX)