This video will provide detailed protocols for in vitro shape experiments to determine the secondary structure of RNA sequences of interest in the presence of an RNA targeting small molecule. In vitro shape is a cost effective method to understand the dynamics of each nucleotide on an amino-sized RNA element which is usually less than 200 nucleotides. After preparing the RNA template as described in the manuscript, radio label reverse transcription primers by adding a gamma 32P ATP, reversed transcription primer.
10XPNK reaction buffer, polynucleotide kinase and RNA's free water in a 1.7 milliliter microcentrifuge tube in a total volume of 20 microliters. Then incubate at 37 degrees Celsius for one hour. You then activate the samples for 20 minutes since 65 degrees Celsius and then filter the samples in a desalting column and centrifuge at 1000 times G.Add 25 microliters of 2XTBE urea sample buffer and mix.
Lower these labeled products into a 10%acrylamide gel and make sure to avoid bleeding the radioactivity into the top reservoirs. Run the gel at 20 watts for one hour. After the run, remove the glass plates of the gel cassette.
Lay the gel on the piece of plastic wrap. Fold the plastic onto the top of the gel to make an air-tight sandwich. Place the gel sandwich on a a calcium-tungstate phosphor screen and expose with an imagining instrument.
Image the gel again with regular light to know where the edges of the gel are. Use an image-processing software to overlay the two images and overlay the light and dark images. Then make the top image slightly opaque in order to see both images simultaneously.
Make sure the printed image has the same size as the actual gel. Then align the gel on the printed image. Lastly, use a sterilized razor blade to cut the desired bends out of the gel and place each piece of gel in a separate 1.7 milliliter microcentrifuge tube.
To recover the DNA from the gel slice by passive elution, add 0.3 milliliters of elution precipitation mix to each 1.7 milliliter tube. Incubate the tubes in a radioactive-safe container on a rotator at four degrees Celsius for 16 hours. After incubation, transfer the liquid into a new 1.7 milliliter tube.
Add 2.5x ice cold 200 proof ethanol and invert the tubes six times to mix the liquid. Incubate the tubes at minus 80 degrees Celsius for a minimum of one hour. After centrifuging at 10, 000g and four degrees Celsius for 10 minutes, remove the supernatant carefully by pipetting.
Add one milliliter of 80%ethanol to wash the pellet. Vortex for 15 seconds and centrifuge again under the same conditions. After removing the supernatant by pipetting, air-dry the DNA pellet for five minutes.
Add 50 microliters of RNA's free water and mix by pipetting up and down 10 times. Normalize the DNA concentration to about 100, 000 counts per minute per microliter in a scintillation counter. To prepare four samples for chemical modification, add eight picomoles of RNA in 32 microliters of 0.5 XTE buffer to a 1.7 milliliter microcentrifuge tube.
Heat at 80 degrees Celsius for two minutes and snap cool on ice for at least one minute. Then add eight microliters of 5x folding mix, mix an allocate nine microliters of this RNA mixture into each of the four PCR tubes, labeled from one to four. Add one microliter of 10%DMSO into the tube marked as one, one microliter of concentrated small molecule solution into tube two, one microliter of diluted small molecule solution into tube three and one microliter of water into tube four.
Incubate all PCR tubes at 37 degrees Celsius in a thermal cycler for 30 minutes. Directly before the two prime OH modification reaction, dilute one microliter of two molar NAI stock solution in three microliters of DEPC-treated water to yield a 0.5 molar working solution. Immediately add one microliter of this solution into tubes one, two and three.
Add one microliter of 25%DMSO into tube four and incubate in a thermal cycler at 37 degrees Celsius for 15 minutes. Prepare four fresh 1.7 milliliter microcentrifuge tubes and add 100 microliters of elution precipitation mix and two microliters of glycogen to each tube. Then into each of these tubes transfer all the liquid from the corresponding PCR tubes.
Add 0.34 milliliters of ice-cold 200 proof ethanol into each tube and mix by vortexing for five seconds. Place the tubes in a minus 80 degrees Celsius freezer for at least one hour. Then centrifuge the tubes for 15 minutes at 14, 000 times G at four degrees Celsius.
Without disturbing the RNA pellet, remove the supernatant from each tube. Add 0.5 milliliters of 80%ethanol per tube to wash the RNA pellet and vortex for 15 seconds. After centrifuging at the same conditions again, remove the supernatant and air-dry the RNA pellet for five minutes.
Add nine microliters of RNA's free water and pipe that up and down 10 times to mix. Transfer the modified RNA into four new PCR tubes and label them one to four as in the previous step. Add two picomoles of RNA in seven microliters of DEPC-treated water to each of four additional PCR tubes and label them with numbers five to eight.
Add two microliters of the recovered radiolabeled primer to each of the eight PCR tubes marked one to eight. After heating at 65 degrees Celsius for five minutes, immediately cool down to four degrees Celsius in the thermal cycler. Add four microliters of 5x RT buffer, one microliter of 0.1 molar DTT and one microliter of DNTP mix to each of the tubes.
Then add two microliters of DEPC-treated water to tubes one to four. Add four microliters of five millimolar ddATP to tube five, four microliters of five millimolar ddTTP to tube six and four microliters of five millimolar ddCTP to tube seven and one microliter of five millimolar ddGTP and three microliters of DEPC-treated water to tube eight and pipette four times to mix. After heating all of the PCR tubes at 52 degrees Celsius for one minute in a thermal cycler, add one microliter of reverse transcriptase to each tube and pipe it up and down four times to mix.
Incubate the reactions at 52 degrees Celsius for 20 minutes in the thermal cycler. To hydrolyze the RNA, add one microliter of five molar sodium hydroxide into each of the tubes and heat them at 95 degrees Celsius for five minutes. Then add five microliters of one molar hydrochloric acid to neutralize the reaction and repeat the ethanol precipitation as previously described.
After air-drying the DNA pellet for five minutes, add 10 microliters of water and 10 microliters of 2xTBE urea sample loading buffer in 1.7 milliliter tubes and pipe it up and down 10 times to redissolve. Heat the samples at 70 degrees Celsius for five minutes, then place the tubes on ice, before loading onto the gel. Load 10 microliters of the sample that has been placed on ice onto 8%polyacrylamide sequencing gel.
Run the gel at 65 watts for two hours or until the bottom dye reaches the 3/4ths of the gel. Remove the spacer and gently insert a spatula to remove the top siliconized glass plate. Cover the gel with a piece of large filter paper and gently press to allow the gel to adhere to the filter paper.
Starting from the bottom end, lift the filter paper and remove the gel from the glass plate together with the paper. After transferring the gel to a wrapping paper as previously done, dry it at 70 degree Celsius for one hour with a vacuum, making sure to use several filter papers between the gel and the dryer. Transfer the gel sandwich onto a photo-bleached phosphor storage screen cassette and expose the radioactivity of the gel to the screen for four to 16 hours.
Finally, place the phosphor storage screen on an imaging device and scan with medium resolution for approximately 30 minutes. After exposing polyacrylamide sequencing gel to the phosphor storage screen, a successful shape experiment showed a single and most intense bend at the top of the gel and bends throughout the gel at single nucleotide resolution without smear. The gel also showed that the intensity of some bends changed in the presence of small molecules, indicating changes of base pairing strength.
A common problem in page analysis is that a smear region known as the salt front may appear in the middle of the gel, probably due to high concentration of salt, DMSO or other unwanted substance in the loading sample. That can be avoided by removing the unwanted substance by ethanol precipitation. A homogenous RNA template is preferable for in vitro shape.
We use ribosomes at both five prime and three prime ends to obtain such an RNA. In-cell shape can also be performed to obtain RNA secondary structure in the cellular context. Remember, in vitro shape may not recapitulate in vivo RNA binding protein interactions.
In vitro shape is a powerful method for detecting changes in RNA structure due to the presence of a small molecule. This is done by quantifying changes in the patterning strength of reverse transcriptase stops. Be cautious that all experiments described in this video be performed in an authorized radioactive workplace with appropriate personal protection and plexiglass shielding.
