Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Mass spectrometry has been widely adopted for various applications. The methodology in this article provides the unequivocal characterization of RNA fragments obtained from an in vitro enzymatic process. Advantages of this technique include that the molecule iron peak is obtained, that the experiments can be carried out with small amounts of material for in vitro applications, and that the protocol can be accomplished with minimal training.

The described methodology can be widely applied to study processes involving DNA, RNA, or proteins. Examples include biochemical transformations such as cross information, identification of chemical modifications, or other biopolymer interactions and reactivity. One difficulty can occur from the accessibility to a MALDI spectrometer or core facilities that offer this service.

Another aspect is establishing the detection limit which may be higher or lower depending on the instrument. To begin, turn on the UV-Vis spectrophotometer, then click on the PerkinElmer UV WinLab icon to open the instrument operation control window. Turn on the Peltier temperature controller spectrophotometer using the switch located to the instrument’s right and click on Scan-Lambda 365, present under Base Methods.

Another screen will open and prompt the user to ensure that the cell holders are empty. Click on OK and allow the instrument to conduct its system checks. Now, adjust the scanning parameters by selecting Data Collection.

In the new window under Scan Settings, change the start to 350 nanometers and the end to 215 nanometers. Activate the Peltier by selecting the plus to the left of the accessory. Then click on Multiple Cell Peltier, change the temperature degrees Celsius to 25, and click on Peltier On.Navigate to the sample info tab and enter the number of samples, names, and cell position desired.

Click on Autozero and insert the cuvette containing the background solution. Click on Start and insert the cuvette in the desired cell and ensure that the cuvette window is oriented parallel to the front face of the instrument. Now, click OK to begin the first scan at 25 degrees Celsius.

For measurements at higher temperatures, click on Multiple Cell Peltier, change the temperature to the desired sample temperature and repeat the scanning. Click on File, Export and choose the desired file location and select XY Data to obtain a text file. To perform 5’phosphorylation of RNA, prepare a solution in a 0.6-microliters micro centrifuge tube by adding 33.5-microliters RNase-free water, five microliters of solution A, six microlites of 10 millimolar adenosine triphosphate, one microlite of 200 micromolar RNA aqueous solution, and 4.5-microliters of polynucleotide kinase and mix the solution gently.

Incubate the reaction mixture at 37 degrees Celsius for 45 minutes by placing it in a water bath. Then inactivating the enzyme by placing the reaction tube in a heat block, preheated at 65 degrees Celsius for 10 minutes. Allow the solution to cool to room temperature to yield a final 5’phosphorylated RNA solution.

After incubation, use 50-microliters of this solution, add five microliters of solution B, and five microliters of 1-femtomole solution of Xrn-1 to perform RNA hydrolysis. Incubate the reaction tube at room temperature for two hours. Transfer this reaction mixture to a 10-kilodalton pore-sized centrifugal device and filter the enzymes by centrifuging for 10 minutes at 700 times G.Transfer the filtrate into a 0.6-milliliter centrifuge tube.

Then, add 20-microliters of RNase-free water to wash the residual RNA on the centrifugal tube, followed by centrifugation. Finally, combine this filtrate with the resultant filtrate obtained previously by centrifugation in 10-kilodaltons pore-sized centrifugal device, and freeze the solution at zero degrees Celsius or ship for analysis. To desalt and concentrate the sample, use commercially available cation exchange C18 pipette tips loaded with a bed of C18 chromatography media at its end.

For this, position the 10-microliter pipette tip onto a 10-microliter pipette and wash this tip twice with 50%aqueous acetyl nitrile solution. Discard the used volumes into a separate waste tube each time and equilibrate the C18 tip with 10-microliters of 0.1%aqueous trifluoroacetic acid solution two times. Manually remove the C18 tip from the pipette and secure it onto a 200-microliter pipette containing a P200 pipette tip.

Then immerse the C18 tip into the resulting solution prepared during hydrolysis of oligonucleotides of RNA by Xrn-1 and aspirate release the solution through the C18 tip 10 times. Now, detach the C18 tip from the P200 pipette, position it down to a 10-microliter pipette and wash the C18 tip using an aqueous solution of 0.1%trifluoroacetic acid solution twice. Discard the used volumes into a separate waste tube each time.

Wash the C18 tip with 10-microliters RNase-free water two times. For spotting onto the MALDI plate, elute the RNA oligonucleotide from the C18 tip by immersing the sample into the desired matrix and dispensing the solution back into the tube 10 times. Pipette out this solution onto two separate spots of one microliter each on the plate and allow spots to air dry.

The concentration of RNA used in this work was determined via UV-Visible spectroscopy recorded at 90 degrees Celsius to avoid erroneous readings arising from the potential formation of the secondary structures. The overall sequence of events included two parts. One, the efficient 5’phosphorylation of RNA, evidenced by the appearance of only one peak corresponding to the expected product.

And two, the stalling fragment arising from the treatment of the sample mixture with Xrn-1. The same results were obtained using a different sequence that contained two oxidative lesions. Potential changes to consider include eliminating the use of the filtering device or considering different matrices for MALDI spotting.

For example, sinapinic acid or picolinic acid. The RNA fragments were obtained from an enzymatic process. This process was established by coupling MALDI, electrophoretic analysis, solid-phase synthesis, and circular dichroism.

The described protocol has a potential to be used in various fields, from research in the chemical sciences to applications in the biomedical field.