The RNA SELEX (Systematic Evolution of Ligands by EXponential enrichment) protocol is designed to select RNA aptamers that bind to a specific target molecule. RNA SELEX is similar to DNA SELEX, but with additional steps to transcribe RNA from a DNA library and manage RNA’s inherent instability. Below is a general protocol for selecting RNA aptamers using SELEX:
Materials Required
DNA library: A pool of DNA molecules with a randomized region flanked by fixed sequences (for primer binding), typically containing a T7 promoter for transcription.
T7 RNA polymerase: For transcription of RNA from the DNA library.
Target molecule: The molecule for which RNA aptamers will be selected (e.g., proteins, small molecules, or cells).
Binding buffer: Typically contains salts (e.g., PBS or Tris buffer) and ions (e.g., Mg²⁺) necessary for RNA folding and binding.
Nuclease-free water and reagents: To avoid degradation of RNA by RNases.
Reverse transcriptase: For converting RNA back to cDNA.
PCR reagents: For amplifying the cDNA after reverse transcription.
RNase inhibitors: To protect RNA during the process.
Column or magnetic beads: For separating bound RNA from unbound RNA.
General RNA Aptamer SELEX Protocol
- Initial DNA Library Preparation:
Design a DNA library with a randomized region (usually 20–80 nucleotides) flanked by fixed sequences for PCR amplification and transcription initiation.
The library should include a T7 promoter sequence at the 5′ end for transcription by T7 RNA polymerase.
Example DNA sequence:
`5’-TAATACGACTCACTATAGG-[random sequence]-GGAGGACGATGCGTC-3’`
The T7 promoter sequence (`TAATACGACTCACTATAGG`) allows for in vitro transcription.
- In Vitro Transcription to RNA:
Use T7 RNA polymerase to transcribe RNA from the DNA library.
Incubate the DNA library with T7 RNA polymerase, NTPs (ATP, GTP, CTP, and UTP), and transcription buffer to synthesize the RNA library.
The resulting RNA pool will contain random regions capable of folding into various 3D structures.
- Incubation with the Target Molecule:
Incubate the RNA library with the target molecule in a binding buffer (containing necessary salts and ions like Mg²⁺ to facilitate RNA folding).
Allow sufficient time (typically 30 minutes to 1 hour) for RNA aptamers to bind to the target.
Binding temperature is usually room temperature or 37°C, depending on the target.
- Separation of Bound and Unbound RNA:
After incubation, separate the bound RNA from unbound RNA.
For small molecules or proteins: Use methods like filtration, affinity columns, or magnetic beads for separation.
For cell SELEX: Wash cells to remove unbound RNA and collect the RNA bound to the target cells.
Increase the stringency of washing steps over subsequent rounds to remove weakly bound or nonspecific sequences.
- Elution of Bound RNA:
Elute the bound RNA using heat, increased salt concentrations, or changes in pH to disrupt RNA-target interactions.
Careful handling is necessary to avoid RNA degradation by RNases.
- Reverse Transcription (RT) of Eluted RNA:
Use reverse transcriptase to convert the eluted RNA into complementary DNA (cDNA).
This cDNA will serve as a template for amplification in the next step.
- PCR Amplification of cDNA:
Amplify the cDNA using PCR with primers complementary to the flanking regions of the library.
Verify successful amplification by running the PCR product on an agarose or polyacrylamide gel.
The amplified cDNA represents the enriched pool of sequences that bound to the target.
- In Vitro Transcription of PCR Product:
After amplification, use T7 RNA polymerase again to transcribe the cDNA back into RNA for the next round of SELEX.
This newly synthesized RNA will be enriched for sequences that have a higher affinity for the target molecule.
- Repetition of SELEX Rounds:
Repeat steps 3 through 8 for several rounds (typically 8–15 rounds), increasing the stringency in each round to enrich for RNA sequences that bind tightly and specifically to the target.
Increased stringency: Reduce target concentration, increase the number of washes, or shorten the incubation time to select for higher-affinity aptamers.
- Monitoring the Enrichment Process:
Monitor the binding affinity and specificity of the enriched RNA pool after several rounds using techniques like:
Electrophoretic Mobility Shift Assay (EMSA): To observe RNA-target interactions.
Surface Plasmon Resonance (SPR): To measure binding kinetics.
Fluorescence-based assays: If the target or RNA aptamer is fluorescently labeled.
Assess binding strength by measuring dissociation constants (Kd) to ensure that the aptamers are becoming more specific and tightly bound to the target.
- Cloning and Sequencing of Final Pool:
After the final round of SELEX, clone the enriched pool of cDNA into a suitable vector and sequence individual aptamers.
Analyze the sequences to identify aptamers with unique or recurring motifs.
Group similar sequences into families based on sequence similarity.
- Characterization of Selected RNA Aptamers:
Test individual aptamers for their binding affinity and specificity to the target.
Measure the dissociation constant (Kd) to determine the strength of the RNA-target interaction.
Validate specificity by testing aptamers against similar targets or control molecules.
Optional: Negative Selection (Counter-SELEX)
Negative selection involves incubating the RNA library with non-target molecules or cells to remove nonspecific binders.
Unbound RNA sequences from negative selection are then used for the next round of SELEX with the target.
This improves specificity by eliminating RNA sequences that bind to unwanted targets.
Notes
Target diversity: RNA SELEX can be performed against various targets, including proteins, small molecules, ions, or whole cells.
Aptamer modifications: After SELEX, RNA aptamers can be chemically modified (e.g., 2′-fluoropyrimidine, 2′-O-methyl modifications) to enhance their stability, especially for in vivo applications.
Summary of RNA SELEX Steps:
- Design and synthesize a DNA library with a T7 promoter.
- In vitro transcription of the DNA library into RNA.
- Incubation of RNA library with the target.
- Separation of bound and unbound RNA.
- Elution of bound RNA.
- Reverse transcription of RNA to cDNA.
- PCR amplification of cDNA.
- Transcription of amplified DNA to generate a new RNA pool.
- Repeat selection rounds with increasing stringency.
- Clone, sequence, and characterize selected RNA aptamers.
This general RNA SELEX protocol can be adapted based on the specific target, desired stringency, and application.