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

1. Protein binding reaction and separation of bound RNA

1. Carry out binding of protein and RNA in 10 mM Tris-HCl, pH 7.5 in a volume of 100 µL by adding the following ingredients to these final concentrations: 50 mM KCl, 1 mM DTT, 0.09 µg/µL bovine serum albumin, 0.5 units/µL RNasin, 0.15 µg/µL tRNA, 1 mM EDTA, and 30 µL of appropriate recombinant protein (PTB) concentration. Add RNA from the appropriate pool.
   NOTE: The splicing factor U2AF65 typically binds to the polypyrimidine-tract/3' splice sites of model introns with a binding affinity (equilibrium dissociation constant or K_d) of approximately 1–10 nM. Therefore, the first two rounds of binding used protein concentration 10-fold above the K_d for U2AF65; for SXL and PTB proteins, the starting concentration in this range was only our best guess. This ensured that desired RNA species could bind, although lower affinity sequences were also potentially bound. In rounds 3 and 4 (transcription, binding, and amplification), the protein concentration was reduced three-fold. This was done to successfully eliminate low-affinity RNA species.
2. Place the tubes containing the binding reactions for about 30 min at 25 °C in a temperature block (or on ice).
3. Fractionate the bound RNA from the unbound RNA for the first 4 rounds of selection-amplification using the following steps.
   1. Filter the sample (100 µL) at room temperature through a nitrocellulose filter attached to a vacuum manifold.
      NOTE: The RNA-protein complex, but not the unbound RNA, remains on the filter.
   2. Chop the filter with retained RNA into fragments with a sterile razor blade; insert these into a centrifuge tube. Recover RNA by tumbling the tube gently for a minimum of 3 h (or overnight) with filter pieces immersed in the proteinase K (PK) buffer.
   3. Deproteinize the RNA sample by vortexing it in the presence of an equal volume of phenol-chloroform (1:1) and then of chloroform. Recover the aqueous phase each time by centrifuging the sample at high speed for 5 min at room temperature.
   4. Mix it with sodium acetate (0.1 volume of 3.0 M, pH 5.2) and ethanol (2–3 volumes of absolute ethanol, 200 proof). Leave the tube in a -80 °C freezer for 30 min, centrifuge it at high speed for 10 min, and, following the washing and drying steps, solubilize the RNA in water treated with DEPC.
4. Separate the protein-bound RNA fractions from the unbound fractions for the last 2 rounds (rounds 5 and 6; transcription, binding, and amplification) as follows. Reduce the protein concentration in the binding reaction (step 1.1) by a further three-fold for additional selection pressure to enrich high-affinity binding sequences and preferentially remove low-affinity sequences.
   1. Pre-cast a native polyacrylamide gel (5% with 60:1 acrylamide:bis-acrylamide ratio) in 0.5x TBE buffer (Tris-Borate-EDTA) prior to setting up the above RNA:protein-binding (step 1.1) reaction. Electrophorese this gel in a cold room (4 °C) by applying 250 V for 15 min.
   2. Pipette the above RNA:protein binding reactions (step 1.1) into different wells of this gel.
      NOTE: The protein is stored at -80 °C and diluted prior to use in 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 8.0, 20% glycerol, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.05% NP-40, and 1 mM dithiothreitol (DTT). The addition of 0.5–1.0 mM protease inhibitor phenylmethane sulfonyl fluoride (PMSF) is optional. In the binding reaction, this buffer contributes about 6% glycerol, which allows the direct loading of samples into the wells without the need for mixing them with a separate gel-loading buffer.
   3. Fractionate the bound RNA from the unbound RNA using gel electrophoresis in a cold room (4 °C) at 250 V for 1 to 2 h. This process is also known as gel mobility shift assay.
      NOTE: Duration of electrophoresis varies depending on features of the RNA and protein used for binding.
   4. 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.
   5. Incubate the crushed gel slice in the PK buffer used for elution for 3 h or overnight.
   6. Vortex the eluted RNA sample vigorously first with phenol-chloroform and then with chloroform.
   7. Mix sodium acetate and ethanol with the aqueous phase after chloroform extraction. Following incubation in a -80 °C freezer, spin at 4 °C for 5–10 min to collect the RNA pellet. Wash the RNA pellet with ethanol and air dry it by leaving the lid of the tube open. Dissolve the RNA in water treated with DEPC.
      NOTE: Switching to the gel mobility shift assay for fractionation allows the elimination of unwanted RNA species that might have been enriched for binding, for example, to the nitrocellulose filter here (or any matrix) used in the initial rounds for fractionation.

2. Reverse transcription and PCR amplification

1. Synthesize cDNA from the dissolved RNA using reverse transcriptase and the reverse primer by incubating the 20 µL reaction (2 µL of 10x RT Buffer, 2 µL of AMV reverse transcriptase, 1 µM reverse primer, 10 µL of RNA, RNase inhibitor optional) at 42 °C for 60 min.
2. Amplify the cDNA using 20–25 PCR cycles. Attach the T7 RNA polymerase promoter to the library by polymerase chain reaction (PCR) containing 1 µM DNA random library template, 1 µM of each primer, 20 mM Tris (pH 8.0), 1.5 mM MgCl2, 50 mM KCl, 0.1 µg/µL acetylated bovine serum albumin, 2 units of Taq polymerase, and 200 µM each of dNTPs (deoxyguanosine, deoxyadenosine, deoxycytidine, and deoxythymidine triphosphate).

3. Transcription and protein binding

1. Repeat the process of RNA synthesis, protein binding, and separation of protein-bound, and unbound fractions.