SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

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.

To bind protein and RNA in a reaction volume of 100 microliters, first prepare 10 millimolar tris-hydrochloride at pH 7.5, containing 50 millimolar potassium chloride, one millimolar DTT, 0.09 micrograms per microliter bovine serum albumin, 0.5 units per microliter RNAsin, 0.15 micrograms per microliter tRNA, and 1 millimolar EDTA. Then, add 30 microliters of recombinant protein PTB and 10 microliters of RNA from the appropriate pool.

Place the tubes containing the binding reactions in a temperature block for about 30 minutes at 25 degrees Celsius. Next, fractionate the bound RNA from the unbound RNA through four rounds of selection and amplification. First, filter the 100-microliter sample at room temperature through a nitrocellulose filter attached to a vacuum manifold. The RNA-protein complex remains on the filter.

With a sterile razor blade, chop the filter into fragments, and insert these into a centrifuge tube. Immerse the filter pieces in proteinase K buffer and place it on a tumbler for a minimum of three hours or overnight to recover the RNA.

To deproteinize the RNA sample, add an equal volume of phenol-chloroform, vortex. And then centrifuge at high speed for five minutes at room temperature. Obtain the aqueous phase, and extract with chloroform again. After that, mix the supernatant with 1/10th volume of 3 molar sodium acetate at pH 5.2 and two to three volumes of absolute ethanol.

Leave the tube in a -80 degrees Celsius freezer for 30 minutes, and then centrifuge it at high speed for 10 minutes. Repeat the washing and drying steps with 70% ethanol, and solubilize the RNA in DEPC-treated water. After the first round of filter-binding assay, carry out three more rounds.

Next, perform two rounds of transcription, binding, and amplification to separate the protein-bound RNA fractions from the unbound fractions. Pre-cast a native 5% polyacrylamide gel with a 60 to 1 acrylamide bisacrylamide ratio in 0.5x TBE buffer. Then, set up the RNA-protein binding reaction.

Place the gel in an electrophoresis device filled with 0.5x TBE buffer in a cold room at 4 degrees Celsius. Apply 250 volts for 15 minutes, and pipette the binding reaction into different wells. Then, further, fractionate the bound RNA from the unbound RNA by running the electrophoresis at 250 volts for one to two hours.

Next, expose the gel to an X-ray film and identify the location of the bound RNA using autoradiography. Cut out the gel slice with the bound RNA and insert it into a tube. Crush the gel slice and incubate in the proteinase K buffer for three hours or overnight. Repeat the extraction with phenol-chloroform and chloroform, as previously described. Synthesize cDNA from the dissolved RNA.

Prepare 20 microliters of reaction, including two microliters of 10x RT buffer, two microliters of AMV reverse transcriptase, 1 microliter of reverse primer, 5 microliters of water, and 10 microliters of the dissolved RNA. Incubate at 42 degrees Celsius for 60 minutes.

Amplify the cDNA using 20 to 25 PCR cycles as previously described. Repeat the process of RNA synthesis, protein binding, and separation of protein-bound and unbound fractions.