Biochemistry
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Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers
Chapters
Summary August 9th, 2022
The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.
Transcript
Studying in Vitro and cellular fluorogenic aptamer kinetics allows the user to evaluate if aptamer dye interactions are fast enough to study a process of interest in real time. The techniques presented allow for quantitative kinetic measurements both inside and outside of the cell, either of which could be relevant depending on the application of the aptamer. The methods presented can be applied to any fluorogenic RNA system, including alternate aptamers and RNA-based biosensors for small molecules.
To begin, set up the RNA renaturation program from the thermocycler. Create the program by selecting create new program"add new phase"and add new step"multiple times to add temperature-specific steps before pressing save"Adjust additional settings according to the program. To renature spinach two and broccoli RNAs, prepare two micromolar stalks of each RNA in double distilled water in a 0.5 milliliter thin walled PCR tube.
Then add equal volumes of two-x renaturation buffer to make one micromolar RNA solution. Place the tubes into the thermocycler. Open the saved renaturation program and press run"Next, set up the plate-reader injector program.
On the fluorescence plate reader, select temperature"and set it to 37 degrees Celsius. Before starting the kinetics, ensure that temperature has equilibrated to this value. Open the plate reader software.
Select settings"then acquisition view"to input the program for kinetic measurements, select loop"for each well. Next, set baseline setting to 60 baseline reads and smart inject settings for 10 microliter injection. Set the fluorescence reads excitation wavelength to 448 nanometers, emission wavelength to 506 nanometers and cartridge to mono.
Set the total read time to 10 minutes with read interval of 0.5 seconds. Set PMT and optics to six flashes per read. Then input loop to next well.
To prepare the plate-reader injector select inject"on the plate reader and supply a waste collection plate when directed. Then select wash"and clean the injection tube following instructions on the plate reader with one milliliter volumes of double distilled water, 75%ethanol, and then distilled water. Next select aspirate"allowing the injector to eject excess liquid.
Then select prime"to prime the injector with two 260 microliters of 100 micromolar D F H B I.To perform one kinetics experiment add 80 microliters of binding buffer master mix to one well of a 96 well clear bottom plate. Then add 10 microliters of renatured RNA. Allow the plate and D F H B I solution in the injector, to equilibrate to 37 degrees Celsius for 15 minutes.
Next, select the well to be analyzed in the software settings under the read area. Then under the home tab, select run"to execute the kinetics program described previously. Repeat this process until all the experiments are complete.
After the run, wash the injector to remove the remaining D F H B I solution from the injection tube as demonstrated previously. To set up the experimental files turn on the flow cytometer and computer. Create a new file within the experiment explorer tab by right clicking the flow cytometer"username.
Then select new experiment"in the dropdown window and when a new window on the computer screen pops up, select experiment type as tubes"and then select okay"In the new experiment file right click the group folder and select add new sample tube"Label the sample tubes for each specific time point and replicate by right clicking on sample"and selecting rename"in the dropdown menu. Next, prepare a diluted cell solution by adding 1.5 milliliters of one X P B S and three microliters of induced cells in AI media in a culture tube. Before adding dye, measure the background fluorescence of the cells to observe the fold turn on over time.
Once the dye is added place the culture tube containing cells in P B S into the sample tube lifter and raise the lifter to the sample injection needle. Then select the proper sample file within the collection panel tab and click record"Once the run is complete lower the sample tube lifter with the culture tube by hand. To initiate a rinse step, to flush the fluidic system, and minimize carryover between each biological replicate sample.
To move to the following sample select the next sample file by clicking the right arrow icon near the sample tube name under the record icon. To measure the fluorescence of cells with dye add 1.4 microliters of concentrated dye stock into one X P B S with cells, to give a final concentration of 50 micromolar D F H B I one T.Secure the culture tube lid and invert three to five times to mix the solution evenly. Next, remove the lid and place the culture tube into the sample tube lifter.
After raising the holder to the sample injection needle, click the record"icon under the proper sample file. Then, begin a timer by pressing start"In vitro kinetics of fluorogenic aptamers spinach two and broccoli, displayed two-phase association kinetics for binding to the D F H B I dye. For both the aptamers, kinetics data were better fitted by two-phase association than one-phase at long measurement times.
The best fit curve determined the rate constants and half-life values for the fast and slow associations. Spinach two in the binding competence state, showed faster turn on than broccoli. The second phase kinetics for both aptamers are similar and may correspond to a common rate-limiting step.
The aptamers kinetic curves are composed of a similar degree of fast associating RNAs. For cells expressing spinach two T RNA, the mean fluorescence intensity or M F I increases immediately at zero time point. Furthermore, cellular fluorescence reached its maximal equilibrated M F I value within five minutes.
In contrast, control cells showed low background fluorescence and no change in M F I values with D M S O addition The methods presented allow researchers to determine how they can make RNA-based sensors in fluorogenic aptamers faster.
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