Name: determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers
Uploaded: 2022-08-09T14:30:00
Description: Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

This work was supported by the following grants to MCH: NSF-BSF 1815508 and NIH R01 GM124589. MRM was partially supported by training grant NIH T32 GM122740.

1. In vitro kinetics experiment
<ol>
	<li>Preparation of DNA templates by PCR
	<ol>
		<li>Set up PCR reaction(s): To prepare PCR reactions, combine the following reagents in a thin-walled PCR tube: 
		33 &#181;L of double-distilled water (ddH2O) 
		10 &#181;L of 5x buffer for high-fidelity DNA polymerase 
		5 &#181;L of 2 mM each deoxyribonucleoside triphosphate (dNTP) 
		0.5 &#181;L of 40 &#181;M forward primer 
		0.5 &#181;L of 40 &#181;M reverse primer<br...

<ol>
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For the in vitro kinetics experiment, the same general protocol can be modified to measure the in vitro kinetics of an RNA-based fluorescent biosensor containing both a ligand-binding and fluorophore-binding domain8. In this case, the RNA should be incubated with the fluorophore prior to measurements upon injecting the ligand in order to obtain ligand response kinetics. If high variability is observed between the replicates, one can troubleshoot by checking that each sample is al...

Fluorogenic reactions are chemical reactions that generate a fluorescence signal. Fluorogenic RNA aptamers typically perform this function by binding a small molecule dye to enhance its fluorescence quantum yield (Figure 1A)1. Different fluorogenic RNA aptamer systems have been developed and consist of specific RNA aptamer sequences and the corresponding dye ligands1. Fluorogenic RNA aptamers have been appended to RNA transcripts as fluorescent tags that enable live cell imaging of mRNAs and non-coding RNAs2,3,4. They have also been placed after promoter sequences as fluorescent reporters of gene expression, similar to the use of green fluorescent protein (GFP) as a reporter, except the reporting function is at the RNA level5,6. Finally, fluorogenic RNA aptamers have been incorporated into RNA-based fluorescent biosensors, which are designed to trigger the fluorogenic reaction in response to a specific small molecule. RNA-based fluorescent biosensors have been developed for live cell imaging of various non-fluorescent metabolites and signaling molecules7,8,9,10,11.
There is growing interest in the development of fluorogenic RNA aptamers to visualize dynamic changes in RNA localization, gene expression, and small molecule signals. For each of these applications, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro kinetics for fluorogenic RNA aptamers Spinach212&#160;and Broccoli13 using a plate reader equipped with a sample injector and to determine the cellular turn-on kinetics for Spinach2 expressed in Escherichia coli using a flow cytometer. These two RNA aptamers were chosen because they have been applied to study RNA localization2,3,4, they have been used in reporters5, 6 and biosensors7,8,9,10,11, and the corresponding dye ligands (DFHBI or DFHBI-1T) are commercially available. A summary of their in vitro properties determined in the literature is given in Table 14,13,14, which informed the protocol development (e.g., the wavelengths and dye concentrations used). These results demonstrate that the fluorogenic reactions affected by RNA aptamers are rapid and should not impede accurate measurements for the desired cell biological applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP160500</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis equipment</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1A-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha D-(+)-lactose monohydrate</td>
			<td>Thermo Fischer Scientific</td>
			<td>18-600-440</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber 1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fischer Scientific</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate ((NH4)2SO4)</td>
			<td>Sigma-Aldrich</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow cytometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24861</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune 1x Focusing Fluid</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24904</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Shutdown Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>A24975</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Performance Tracking Beads</td>
			<td>Thermo Fischer Scientific</td>
			<td>4449754</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Wash Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>&#160;J24974</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>C3416</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorine Bleach</td>
			<td>Amazon</td>
			<td>B07J6FJR8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar 96-well plate</td>
			<td>Daigger Scientific</td>
			<td>EF86610A</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Tubes, 12 mm x 75 mm, 5 mL with attached dual position cap</td>
			<td>Globe Scientific</td>
			<td>05-402-31</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI</td>
			<td>Sigma-Aldrich</td>
			<td>SML1627</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI-1T</td>
			<td>Sigma-Aldrich</td>
			<td>SML2697</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose (anhydrous)</td>
			<td>Acros Organics</td>
			<td>AC410955000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA loading dye</td>
			<td>New England Biolabs</td>
			<td>B7025S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes (2.0 mL)</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs: dATP, dCTP, dGTP, dTTP</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, pH 8.0</td>
			<td>Gibco, Life Technologies</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter-tip micropipettor tips</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM12635, AM12648, AM12655, AM12665</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>FlowJo v10 Software</td>
		</tr>
		<tr>
			<td>Fluorescent plate reader with heating control</td>
			<td>VWR</td>
			<td>10014-924</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electrophoresis power supply</td>
			<td>Thermo Fischer Scientific</td>
			<td>EC3000XL2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen AM95010</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM95010</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Analysis software from Academic Group License&#160;</td>
		</tr>
		<tr>
			<td>Heat block&#160;</td>
			<td>Thomas Scientific</td>
			<td>1159Z11</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Inorganic pyrophosphatase</td>
			<td>Sigma-Aldrich</td>
			<td>I1643-500UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Molecular Weight DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N3233L</td>
			<td>Supplied with free vial of Gel Loading Dye, Purple (6x), no SDS (NEB #B7025).</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Fisher Scientific</td>
			<td>MFCD00011110</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge with temperature control</td>
			<td>Marshall Scientific</td>
			<td>EP-5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettors</td>
			<td>Gilson</td>
			<td>FA10001M, FA10003M, FA10005M, FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor tips</td>
			<td>Sigma-Aldrich</td>
			<td>Z369004, AXYT200CR, AXYT1000CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore water filter with BioPak unit</td>
			<td>Sigma-Aldrich</td>
			<td>CDUFBI001, ZRQSVR3WW</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow micropipettor pipette tips</td>
			<td>DOT Scientific</td>
			<td>RN005R-LRS</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP39920</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR clean-up kit</td>
			<td>Qiagen</td>
			<td>28181</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers and templates</td>
			<td>Integrated DNA technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler for thin-walled PCR tubes</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler for 0.5 mL tubes</td>
			<td>Techne</td>
			<td>5PRIME/C</td>
			<td></td>
		</tr>
		<tr>
			<td>pET31b-T7-Spinach2 Plasmid</td>
			<td>Addgene</td>
			<td>Plasmid #79783</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase&#160;</td>
			<td>New England Biolabs</td>
			<td>M0530L</td>
			<td>Purchase of Phusion High-Fideldity Enzyme is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, and MgCl2 and DMSO solutions.</td>
		</tr>
		<tr>
			<td>Polyacrylamide gel electrophoresis gel comb, C.B.S. Scientific</td>
			<td>C.B.S. Scientific</td>
			<td>VGC-1508</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide gel electrophoresis equipment</td>
			<td>C.B.S. Scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Genesee Scientific</td>
			<td>38-101</td>
			<td></td>
		</tr>
		<tr>
			<td>rNTPs: ATP, CTP, GTP, UTP</td>
			<td>New England Biolabs</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Short wave UV light source</td>
			<td>Thermo Fischer Scientific</td>
			<td>11758221</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic, anhydrous</td>
			<td>Thermo Fischer Scientific</td>
			<td>S375-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>SoftMax Pro 6.5.1 (platereader software) obtained through Academic Group License</td>
		</tr>
		<tr>
			<td>Sterile filter units</td>
			<td>Thermo Fischer Scientific</td>
			<td>09-741-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>Thermo Fischer Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer for agarose gel electrophoresis</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9869</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>TRIS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone (granulated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea-PAGE Gel system&#160;</td>
			<td>National Diagnostics</td>
			<td>EC-833</td>
			<td></td>
		</tr>
		<tr>
			<td>UV fluorescent TLC plate</td>
			<td>Sigma-Aldrich</td>
			<td>1.05789.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Vis spectrophotometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>ND-8000-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Thermo Fischer Scientific</td>
			<td>2215415</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Sigma-Aldrich</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract (Granulated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>BP9727-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s&#8722;1 and 0.35 s&#8722;1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

In vitro kinetics 
The sequences of the DNA templates and primers, which are purchased as synthetic oligonucleotides, are shown in Table 2, and the reagent recipes are shown in Supplementary File 1. PCR amplification is used to scale up the amount of DNA template with the T7 promoter, which is required for the subsequent in vitro transcription (IVT) reaction. In addition, PCR amplification can be used for two purposes in the same r...

Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

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.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

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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