The overall goal of this toeprinting analysis is to observe the translation initiation complex formation on mammalian mRNAs. This method can help answer key questions in the field of mRNA translation, such as IRES mediated translation initiation. The main advantage of this technique is that one can perform detailed analysis of translation initiation complex formation on a specific mRNA.

Assemble the transcription reactions to transcribe RNA from the PCR amplified DNA templates, encoding the XIAP IRES RNA or its mutants. Next, add two units of RNase-free DNase to remove the DNA template to each 20 microliter reaction of RNA. Incubate at 37 degrees Celsius for 30 minutes.

Then, dilute the DNase treated RNA with nuclease free water to a final volume of 110 microliters. Next, add 110 microliters of acid phenol to begin RNA purification. Vortex the samples for five seconds and centrifuge at 20, 000 x g for three minutes at room temperature.

It's critical to dilute the RNA or CDNA prior to phenol extraction. If the volume is too low, it's gonna be difficult to physically recover the aqueous phase and the final yield will be low. Then, carefully transfer 100 microliters of the RNA-containing aqueous phase to a new microcentrifuge tube.

Add 10 microliters of 3 M sodium acetate, pH 5.5, and mix the samples. Then, add three volumes of 100%ethanol to precipitate RNA and vortex the samples for five seconds. Incubate overnight at 20 degrees Celsius.

The next day, centrifuge the samples at 20, 000 x g for 30 minutes at four degrees Celsius and discard the supernatant. Then, wash the pellet with 500 microliters of ice cold 70%ethanol and repeat the centrifugation. Carefully aspirate as much supernatant as possible without dislodging the pellet.

Then, air dry the pellet for five to 10 minutes. Resuspend the pellet in nuclease-free water to a final concentration of 0.5 micrograms per microliter. To begin the toeprinting assay, add 15 microliters of non-nuclease treated rabbit reticulocyte lysate to each 1.5 milliliter microcentrifuge tube, which represent the wild-type XIAP IRES RNA and its mutants.

Add RNase inhibitor, ATP, and GMP-PNP to each tube. Incubate at 30 degrees Celsius for five minutes. After this, add one microliter of the respective RNA to each of the tubes and incubate at 30 degrees Celsius for an additional five minutes.

During the incubation period, add DTT and spermidine to the toeprinting buffer. Then, add 22 microliters of toeprinting buffer to each of the tubes and incubate at 30 degrees Celsius for three minutes. Next, add 0.5 microliters of IR dye labeled toeprinting primer and incubate the reaction on ice for 10 minutes.

Subsequently, add two microliters of dNTPs, two microliters of magnesium acetate, and one microliter of avian myeoloblastosis virus reverse transcriptase. Bring the final volume to 50 microliters with toeprinting buffer. Then, allow primer extension to occur for 45 minutes at 30 degrees Celsius.

It's also critical to add magnesium acetate to the toeprinting reaction, without which, the reverse transcriptase won't function optimally. After this, add 200 microliters of nuclease-free water. Add 250 microliters of 25:24:1 phenol:chloroform:isoamyl alcohol, pH 8.0, to begin the purification of cDNA.

After vortexing the samples for five seconds, centrifuge the samples at 20, 000 x g at room temperature for three minutes. Next, carefully transfer the aqueous phase containing the DNA to a new microcentrifuge tube. Then, add three volumes of 100%ethanol to precipitate the DNA and vortex for five seconds.

Incubate overnight at 20 degrees Celsius. The next day, centrifuge the tubes at 20, 000 x g for 30 minutes at four degrees Celsius and discard the supernatant. Then, wash the DNA pellet with 500 microliters of ice cold 70%ethanol and centrifuge at 20, 000 x g for 15 minutes at four degrees Celsius.

Aspirate as much as supernatant as possible, taking care not to dislodge the pellet. Then, air dry the pellet for five to 10 minutes. Dissolve the DNA pellet in six microliters of nuclease-free water.

Then, add three microliters of formamide loading dye. Use a sequencing kit to prepare a dideoxy nucleotide sequencing ladder with the toeprinting primer and the DNA construct encoding the XIAP IRES RNA. Then, mix six microliters of each sequencing reaction with three microliters of the formamide loading dye.

After cleaning and assembling the plates, pour the gel, and insert the comb. Following the polymerization of the gel, assemble the sequencing apparatus and fill the reservoirs with 1x TBE. Then, prerun at 1500 volts for 15 minutes until a temperature of 55 degrees Celsius is achieved.

Heat all of the samples with formamide dye to 85 degrees Celsius for five minutes. Then, load one microliter of each sample on a 6%polyacrylamide sequencing gel and run the gel at 1500 volts for eight hours. Finally, read the bands in realtime with a fluorescent based imager.

This method shows that confirmation of RNA is critical for translation initiation complex formation. The reverse transcription of XIAP mRNA ribosome complex yields typical toeprints 17 to 19 nucleotides downstream of AUG. The 5'PPT mutant shows impaired toeprint formation.

However, when transcribed with the 5'cap, initiation complex formation occurs. Similarly, the 3'PPT mutant also shows impaired toeprint formation. However, transcription with the 5'cap leads to initiation complex formation.

The double PPT mutant harboring both the 5'PPT and 3'PPT mutations is able to form the initiation complex without the 5'cap. Toeprint formation is strongly impaired for the start codon mutant in the absence of RRL and GMP-PNP and a Poly A tail. The CDNA products are compared with a dideoxy nucleotide sequencing ladder.

Human beta globin, or HBB mRNA, shows no toeprint in the absence of the 5'cap. However, the initiation complex is formed on the capped 5'UTR of HBB, yielding toeprints 16 to 18 nucleotides downstream of AUG. Once mastered, this technique can be done in three days, including preparation of in vitro transcription templates and RNA, if performed properly.

Following this procedure, other methods like structural probing can be performed in order to answer additional questions like the secondary structure of the wild-type or mutant variants of the IRES. After its development, this technique has paved the way for researchers in the field of mRNA translation to explore IRES mediated translation in vitro. After watching this video, you should have a good understanding of how to perform toeprinting of mammalian mRNA and to analyze the formation of translation initiation complexes.

Don't forget that acid-phenol, phenol:chloroform:isoamyl alcohol and acrylamide can be extremely hazardous. Appropriate personal protective equipment should be worn while using these reagents.