SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions
Transkript
Systematic evolution of ligands by exponential enrichment, SELEX, allows the identification of protein-binding RNA sequences.
First, obtain a randomized RNA pool containing radiolabeled RNAs with randomized sequences that fold into highly-specific structures. Incubate with the target protein. The protein binds to RNA molecules with specific sequences and three-dimensional structures, while non-specific RNAs remain unbound.
Pass the mixture through a nitrocellulose filter. Unbound RNA passes through filter pores, while RNA-protein complexes remain retained.
Chop the RNA-protein complex-containing filter into fragments. Repeat RNA-protein interaction by incubating the RNA pool with reduced target protein concentration — allowing only high-affinity sequence binding, while low-affinity sequences fail to bind.
Load this mixture on a polyacrylamide gel and perform electrophoresis. The RNA-protein complexes migrate slowly through the gel compared to low-affinity, unbound RNAs. X-ray gel imaging shows a band shift corresponding to the RNA-protein complex location.
Slice the complex-containing gel portion. Add Proteinase K to the filter fragments and gel slices, digesting the proteins and releasing RNA from the complex. Add phenol-chloroform and centrifuge, separating the RNA from the digested proteins.
Mix the RNA-containing supernatant with sodium acetate and ethanol. Centrifuge to precipitate the RNA. Resuspend in RNase-free water. Reverse-transcribe the RNA to complementary DNA and PCR-amplify the DNA.
Repeat several rounds of filter-based and gel-based separation to obtain a pool of target protein-specific DNA sequences.
