A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

The overall goal of this phosphorothioate approach in RNA is to perform saturation mutagenesis in a single step. This method can help answer key questions in the molecular biology field such as gene regulation. The main benefit of this technique is that in a single step one can accomplish saturation mutagenesis of a protein binding site in RNA.

A key step in this single-step saturation mutagenesis protocol is to dope the DNA template with non-wild type nucleotides within the protein-binding site. First synthesize a T7 primer by chemical synthesis on a DNA synthesizer. Next synthesize a doped oligonucleotide corresponding to the protein binding site.

In this example, the underlined sequence contains the reverse complement of the drosophilas sex-lethal protein binding site, with each doping site represented by an X.Use a ratio of 90%wild type nucleotide to 10%non-wild type nucleotide for each site of doping. The sequence in green is complementary to the T7 primer sequence and is the T7 promoter for in vitro transcription. The next step in this protocol is the synthesis of separate RNAs with each phosphorothioate nucleotide followed by 5-prime-end radiolabeling of the RNA.

Synthesize RNAs for each phosphorothioate in a 20-microliter transcription reaction. To each microcentrifuge tube, add the following:T7 transcription buffer, one micromolar T7 oligonucleotide, one micromolar doped oligonucleotide, 10 millimolar DTT, two millimolar GTP, one millimolar each of ATP, CTP, and UTP, and two units per microliter of T7 RNA polymerase. Add 0.167 millimolar of alpha-thio ATP to one tube and 0.05 millimolar of alpha-thio UTP to the other tube.

Incubate the RNA synthesis reaction mixtures at 37 degrees Celsius for two hours. After two hours, add 2.5 microliters of heat-labile alpha phosphatase and 2.5 microliters of 10X phosphatase buffer to each RNA sample. Incubate at 37 degrees Celsius for 10 to 30 minutes to remove 5-prime phosphates.

Inactivate the alkaline phosphatase enzyme by heating at 80 degrees Celsius for two to five minutes. The remaining steps involve the use of radioactivity and must be performed using appropriate precautions and a Plexiglass shield to protect from radioactivity. To radiolabel the 5-prime end of the dephosphorylated RNA, use one microliter of T4 polynucleotide kinase and one microliter of Gamma P32 ATP in a 10-microliter reaction volume.

Incubate the reaction mixes at 37 degrees Celsius for 30 to 60 minutes. Inactivate the T4 polynucleotide kinase enzyme by heating at 65 degrees Celsius for 20 to 30 minutes. Run the reactions in a 10%denaturing polyacrylamide gel.

Locate RNA on the gel by autoradiography and excise the gel slice containing radiolabeled RNA. Crush the gel slice in a microcentrifuge tube by pressing it against the side with a pipette tip, and then add proteinase K buffer. Rotate the tubes at room temperature from two hours to overnight.

Next centrifuge the gel slurry, collect the buffer solution, and discard the gel. Extract each solution twice with an equal volume of phenol chloroform. After that, extract the solution once with chloroform.

Collect the aqueous phase and add to it 0.1 volume of three-molar sodium acetate pH 5.2, carrier tRNA or glycogen, and 2.5 volumes of ethanol. Keep the samples at minus 80 degrees Celsius for one hour. Centrifuge the samples in a high-speed microcentrifuge at 16, 873 times g for five to 10 minutes.

Remove the buffer ethanol solution carefully without disturbing the RNA pellet. Wash the pellets with 70%ethanol and centrifuge for two to five minutes. Remove the ethanol carefully and air dry the pellets.

Resuspend each pellet in 20 to 50 microliters of DEPC-treated water. Store at minus 20 degrees Celsius until use. The concept of partitioning due to interference is illustrated here.

During the process of protein binding, RNA molecules partition between the protein-bound fraction and the unbound fraction. If a specific nucleotide at a given position interferes with protein binding, it will be preferentially excluded from the protein-bound fraction. Subsequently, iodine is used to cleave the RNAs at the sites of phosphorothioate incorporation.

To begin this procedures, set up the protein-RNA binding reactions with each reaction containing 10 millimolar Tris-HCl, one millimolar DTT, 50 millimolar potassium chloride, 0.5 units per microliter of RNase inhibitor, 0.09 micrograms per microliter of acetylated bovine serum albumin, one millimolar EDTA, 0.15 micrograms per microliter of tRNA, 5-prime-end radiolabeled RNA, and six microliters of an appropriate concentration of the protein. Incubate the protein-binding reactions at 25 degrees Celsius for 20 to 30 minutes. Separate the protein-bound RNA fraction from the unbound fraction by nitrocellulose filter binding.

Apply the binding reaction onto a nitrocellulose filter connected to a vacuum manifold at room temperature. Only the RNA protein complex will be retained on the filter while unbound RNA flows through the filter. Cut the portion of the nitrocellulose filter containing the retained radioactive RNA into smaller pieces to fit into a microcentrifuge tube and add sufficient proteinase K buffer to immerse the filter pieces.

Elute RNA from the filter pieces for two to three hours or overnight. Next, transfer the solution from each tube to a new tube and extract with phenol chloroform and chloroform as demonstrated earlier. Collect the aqueous phase and add to it 0.1 volume of three-molar sodium acetate pH 5.2 and 2.5 volumes of ethanol and incubate in the freezer.

After centrifuging, washing, and drying the RNA as shown earlier, resuspend the RNA in DEPC-treated water. All steps involving iodine must be performed in an exhaust hood. To cleave RNAs at the sites of phosphorothioate incorporation, add to each RNA sample one-millimolar iodine in 20 microliters of DEPC-treated water containing up to 10 micrograms of carrier tRNA.

Incubate at room temperature for five minutes. Precipitate cleaved RNA by adding sodium acetate and ethanol as shown previously. Resuspend cleaved RNA in a loading dye for denaturing gels.

After heating, load the samples in a 15-20%denaturing polyacrylamide gel and separate the RNA fragments by electrophoresis. Expose the polyacrylamide gel to an X-ray film and detect bands in the bound fraction versus total pool using autoradiography. This schematic illustrates the concept of partitioning due to interference.

In lane T, the total RNA fraction, band intensity is approximately equal for all doped positions. In the protein-bound RNA fraction, at positions 1, 4, and 7, the nucleotide has no effect on binding. However, at positions 3 and 6, it interferes with binding and is excluded from the bound fraction.

At position 5, interference is partial. Comparisons of paired lanes for each nucleotide allow for analysis of all four nucleotides. This autoradiograph shows two pairs of lanes from a denaturing gel.

Lane T is total RNA and lane B is protein-bound RNA. The vertical line marks the sex-lethal binding site. For the alpha-thio aline pair, bands 1, 2, 3, and 5 are present in the total RNA fraction but absent or significantly reduced in the bound RNA fraction.

For the alpha-thio uline pair, bands 7 and 8 are relatively less intense in the bound fraction. Residues that are preferentially excluded from the bound fraction are important for protein binding. Once RNA's synthesized and 5-prime-end labeled, this technique can be done in 12 hours if it is performed properly.

While attempting this procedure, it's important to remember that appropriate level of phosphorothioate incorporation and determination of appropriate protein concentration for binding are important considerations. Following this procedure, other methods like appropriate functional assays or in vivo tests can be done to validate the outcome of the mutagenesis approach and understand the biological relevance. This technique provides an alternative to other methods that are either more expensive or more laborious or both.

After watching this video, you should have a good understanding of how to design and synthesize mutant library, perform binding reactions, identify mutant nucleotides in each pool, and analyze quantitatively the effect of non-wild type nucleotides at each position tested. Don't forget that working with polyacrylamide and radioactivity can be hazardous and precautions, such as using gloves, proper exhaust, and radioactive shields should always be taken while performing this procedure.