Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

The protocol is used for saturation mutagenesis of a protein-binding site. If your protein-binding site is not known, it can be identified using this protocol. The technique has relevance for disease diagnosis and therapy.

I believe its full potential in biomedical sciences remains to be fully exploited. This method can be broadly applied to any protein or reaction where desired molecules can be separated from undesired molecules. Make sure that the library is properly randomized, and make sure that during each binding and separation step fold enrichment of desired molecules is high.

It involves several steps and many details that are better demonstrated, rather than explained in words. After watching this video, you should be comfortable with how to prepare a random library, synthesize a starting RNA pool, and perform a binding reaction to separate desired and undesired molecules to obtain high affinity and specific binders. First, synthesize the forward primer and the reverse primer by chemical synthesis on a DNA synthesizer.

Also, synthesize a random library oligonucleotide template with 31 randomized positions. In a PCR tube, mix one-micromolar DNA random library template, one-micromolar forward primer containing T7 RNA polymerase promoter and reverse primer, 20-millimolar Tris at pH eight, 1.5-millimolar magnesium chloride, 50-millimolar potassium chloride, 1-micrograms-per-milliliter acetylated bovine serum albumin, two units of Taq polymerase, and 200 micromolar each of dNTPs. Set up the PCR machine with five cycles of denaturation, annealing, and extension steps, followed by one cycle of extension to attach the promoter to the DNA library.

Set up a 100-microliter transcription reaction containing T7 transcription buffer, one-micromolar random library pool DNA, five-millimolar DTT, two-millimolar GTP, one millimolar each of ATP, CTP, and UTP, and two-units-per-microliter T7 RNA polymerase. Now put on an acrylic glass shield and gloves. Introduce radioactivity by adding 5 microliters or less of alpha-32P UTP into the transcription reaction for autoradiography.

Incubate the reaction mixture in a microcentrifuge tube for two hours at 37 degrees Celsius. To gel-purify RNA, prepare a 10%denaturing polyacrylamide gel. Load samples into the wells of the gel.

Expose the gel to an x-ray film, and identify the location of the transcripts on the gel and cut out the gel slice. Place the gel slice in a centrifuge tube and break into smaller pieces with a homogenizer tip. Add proteinase K buffer to immerse the gel pieces.

Leave the tube on a nutator for at least two hours at room temperature. Then spin in a high-speed microcentrifuge for five minutes at room temperature to remove the gel debris and recover the buffer solution. Add an equal volume of phenol-chloroform into the tube with the supernatant, vortex, and centrifuge.

Repeat the extraction with phenol-chloroform one more time and then with chloroform one more time. Next, mix the aqueous phase of proteinase K buffer with 1/10 volume of three-molar sodium acetate at pH 5.2, 10 micrograms of tRNA or 20 micrograms of glycogen, and two to three volumes of ethanol stored at minus 20 degrees Celsius. Leave the tube at minus 80 degrees Celsius for one hour.

Spin the tube containing the solution for five to 10 minutes at four degrees Celsius in a microcentrifuge. Discard the supernatant carefully, and add 70%ethanol to rinse the RNA pellet. Spin for two to five minutes.

Aspirate the ethanol carefully, and air-dry the RNA pellet. Next, add 50 microliters of water treated with DEPC to solubilize the RNA pellet. Leave the sample at minus 20 degrees Celsius for storage.

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, 09-micrograms-per-microliter bovine serum albumin, 5-units-per-microliter RNAsin, 15-micrograms-per-microliter tRNA, and one-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 1/10 volume of three-molar sodium acetate at pH 5.2 and two to three volumes of absolute ethanol. Leave the tube in a minus 80-degree 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.

Precast a native 5%polyacrylamide gel with 60-to-one acrylamide-bis-acrylamide ratio in 5x TBE buffer. Then set up the RNA-protein binding reaction. Place the gel in an electrophoresis device filled with 5x TBE buffer in a cold room at four 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, one microliter of reverse primer, five 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. To analyze the protein binding, use the gel mobility shift assay or the filter binding assay to determine binding affinity and specificity for selected pools or individual sequences within each pool.

Use autoradiography or a phosphor imager to detect and quantify bands in the bound fraction and unbound fraction. Continue the protocol with cloning and sequence alignment. In this study, a successful selection-amplification was achieved.

The mammalian polypyrimidine-tract binding protein with 300-fold higher protein concentration shows barely detectable binding to the pool zero sequence but high affinity for the selected sequence pool. Selection-amplification successfully identified on RNA-binding proteins with preferred or consensus binding site. Addition of the recombinant PTB, which binds to an upstream three-prime splice site, leads to activation of the alternative or downstream three-prime splice site.

In contrast, addition of recombinant hnRNP C leads to repression of both three-prime splice sites. Addition of the recombinant general splicing factor U2AF65 reversed the hnRNP C-mediated three-prime splice site repression. It's important to remember that a good randomized library and proper binding conditions that give high fold enrichment at each binding step are necessary for success.

Appropriate functional assays and in vivo tests can be used to validate the outcome of this approach. In the field of gene expression, functions of numerous proteins have been studied using this technique. It's important to use gloves and radioactive seal for protection while working with polyacrylamide and radioactivity.