DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

DNAzyme-dependent digestion is a useful method for studying functions of snoRNAs. It is a quick and simple method to analyze site-specific RNA methylation. It requires a short DNA oligonucleotide and basic reagents present in any molecular biology lab.

Begin by designing the DNAzyme for the sequence of interest. Find the RNA sequence or putative methylation site using an appropriate database. For S.cerevisiae snoRNA targets, use the yeast snoRNA database.

To find a methylation site of interest, for example, an snR13-dependent site, select snR13 and make a note of the position of the modified nucleotide. Find the sequence upstream and downstream of the modified nucleotide using an appropriate database, such as Saccharomyces Genome Database. Search for the target gene name.

If using a 10 to 23 DNAzyme assay, select 10 to 15 nucleotides upstream and downstream of the methylation site. If using the 8-17 DNAzyme, select 20 nucleotides. Create complementary sequences of the five-prime and three-prime arms and use them to flank the DNAzyme catalytic sequence on the three-prime and five-prime ends.

Order the DNAzyme as a normal DNA oligonucleotide. Grow yeast strains of interest in an appropriate medium and conditions. When cells reach middle exponential phase, pellet them by centrifuging at 1, 000 times g for three minutes at four degrees Celsius and discard the medium.

Proceed with RNA isolation according to manuscript directions. Then, resuspend the RNA pellet in 30 microliters of RNase and DNase-free water. Place the tube on ice and measure the RNA concentration on a microspectrophotometer.

To perform DNAzyme digestion using the 10 to 23 DNAzyme, prepare an incubation mix with five micrograms of RNA, 200 picomoles of 10 to 23 DNAzyme, and one-X 10 to 23 incubation buffer in a total volume of 10 microliters. Then, incubate the tubes on a dry heat block set to 95 degrees Celsius for three minutes. Immediately after, place the tubes on ice and leave them there for five minutes.

Briefly spin down the tubes and put them back on ice. Add 20 units of RNase inhibitor and place the tubes on a dry heat block set to 25 degrees Celsius for 10 minutes. Meanwhile, prepare a reaction mixture by combining five microliters of four-X 10 to 23 reaction buffer with four microliters of 300-molar magnesium chloride and one microliter water.

Preheat this reaction mix on a dry heat block set to 37 degrees Celsius. Place the tube with the incubation mix on the 37-degree Celsius dry heat block and add 10 microliters of the prewarmed reaction mix. Incubate the mixture at 37 degrees Celsius for one hour and then, place the tube on ice.

To perform DNAzyme digestion using the 8-17 DNAzyme, prepare a 1.5-milliliter microtube with five micrograms of RNA in a total volume of six microliters. Then, prepare a separate tube with 400 picomoles of the 8-17 DNAzyme. While working, keep both tubes on ice.

Transfer both tubes to a dry heat block set to 95 degrees Celsius and incubate them for two minutes. Move the RNA sample back on ice and spin down the tube with the DNAzyme for five seconds. Incubate the DNAzyme at 25 degrees Celsius for 10 minutes.

While the DNAzyme is incubating, prepare 10 mircoliters of two-X 8-17 reaction buffer and prewarm it to 25 degrees Celsius. Add the prewarmed buffer to the tube with the DNAzyme and then, transfer 14 microliters of this reaction mix to the tube with the RNA, along with 20 units of RNase inhibitor. Incubate the reaction at 25 degrees Celsius for two hours and then, place the tube on ice.

To purify the RNA after DNAzyme digestion, add 350 microliters of water and 400 microliters of chloroform to the reaction tube. Vortex for 30 seconds and centrifuge at 20, 000 times g for five minutes. After centrifugation, transfer the upper phase to a new tube containing one milliliter ethanol, 40 microliters of 7.5-molar ammonium acetate, and one microliter of glycogen.

Flip the tube a few times to mix and incubate either at negative 80 degrees Celsius for two hours or at negative 20 degrees Celsius overnight. Proceed with RNA purification according to manuscript directions and then, re-suspend the RNA pellet in 10 microliters RNase and DNase-free water. Place the RNA tube on ice immediately after purification.

To perform RNA electrophoresis, start by dissolving 1.5 grams of agarose in 127.5-milliliters of double distilled water by heating the mixture in the microwave. Then, add 15 milliliters of 10X MOPS and 7.5 milliliters of 37%formaldehyde to the agarose solution. Add an appropriate amount of gel stain to the agarose solution.

Pour the agarose into the tray and let it cool for 45 minutes. Prepare the RNA samples by combining 10 microliters of the digested and purified RNA with five microliters of sample denaturing buffer and 0.5 microliters of six-X loading dye. Incubate the samples at 70 degrees Celsius for five minutes and then, on ice for five minutes.

Put the prepared gel into the electrophoresis tank and fill the tank with one-X MOPS buffer. Load the entire 15 microliters of the sample onto the gel and run the gel at 80 volts until bromothymol blue reaches 2/3 of the gel length. Analyze the gel with the appropriate imager.

The value of this technique can be demonstrated on an inducible snoRNA transcription system. Inserting an inducible GAL1 promoter upstream of either snR13 or snR47 genes allows for analysis of snoRNA-dependent methylation of 25S ribosomal RNA. When the cells are grown on galactose, the 25S ribosomal RNA is methylated at snoRNA guided sites and remains intact after DNAzyme treatment.

In contrast, when snoRNA expression is shut off due to the lack of galactose, DNAzyme dependent cleavage occurs. Furthermore, no RNA digestion is observed in the wild-type controls since snoRNA expression is galactose independent. The activity of the DNAzyme-dependent cleavage in analysis of our rRNA modifications has been shown recently in the context of snoRNA maturation.

The DNAzyme-dependent assay was used to show that lack of five-prime and pre-snoRNA processing affects methylation levels of 25S and 18S rRNA in Saccharomyces cerevisiae. This protocol requires using phenol and chloroform, which are toxic, and should be handled under a fume hood.