Name: nonradioactive assay to measure polynucleotide phosphorylation of small nucleotide substrates
Uploaded: 2020-05-08T15:00:00
Description: Watch this Scientific Journal Video about Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates at JoVE.com

We thank Dr. Andrew Sikkema and Andrea Kaminski for their critical reading of this manuscript. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS; ZIA ES103247 to R.E.S) and the Canadian Institutes of Health Research (CIHR; 146626 to M.C.P).

1. Preparation
<ol>
	<li>Prepare buffer and reagents.
	<ol>
		<li>Make 1x Reaction Buffer by combining 20 &#181;L of 1 M Tris (pH = 8.0), 40 &#181;L of 5 M sodium chloride, 2.5 &#181;L of 2 M magnesium chloride, 100 &#181;L of 50% (v/v) glycerol, and RNase-free water to reach a total volume of 1 mL.</li>
		<li>Make urea loading dye by combining 4.8 g of urea, 200 &#181;L of 1 M Tris (pH = 8.0), 20 &#181;L of 0.5 M EDTA (pH = 8.0), 0.5 mL of 1% (w/v) bromophenol blue, and RNase-free water to reach a total volume of 10 m...

<ol>
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D., Shuman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+of+T4+polynucleotide+kinase:+an+RNA+repair+enzyme.">Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme.</a> EMBO Journal. 21 (14), 3873-3880 (2002).</li><li>Zhang, Y., Zhao, J., Chen, S., Li, S., Zhao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+microchip+electrophoresis+laser+induced+fluorescence+detection+method+for+the+assay+of+T4+polynucleotide+kinase+activity+and+inhibitors.">A novel microchip electrophoresis laser induced fluorescence detection method for the assay of T4 polynucleotide kinase activity and inhibitors.</a> Talanta. 202, 317-322 (2019).</li><li>Cui, L., Li, Y., Lu, M., Tang, B., Zhang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrasensitive+electrochemical+biosensor+for+polynucleotide+kinase+assay+based+on+gold+nanoparticle-mediated+lambda+exonuclease+cleavage-induced+signal+amplification.">An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification.</a> Biosensors and Bioelectronics. 99, 1-7 (2018).</li><li>Wang, L. J., Zhang, Q., Tang, B., Zhang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Detection+of+Polynucleotide+Kinase+Based+on+Phosphorylation-Directed+Recovery+of+Fluorescence+Quenched+by+Au+Nanoparticles.">Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles.</a> Analytical Chemistry. 89 (13), 7255-7261 (2017).</li><li>Liu, H., Ma, C., Wang, J., Chen, H., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+colorimetric+assay+for+T4+polynucleotide+kinase/phosphatase+activity+and+its+inhibitors+based+on+G-quadruplex/hemin+DNAzyme.">Label-free colorimetric assay for T4 polynucleotide kinase/phosphatase activity and its inhibitors based on G-quadruplex/hemin DNAzyme.</a> Analytical Biochemistry. 517, 18-21 (2017).</li><li>Du, J., Xu, Q., Lu, X., Zhang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+label-free+bioluminescent+sensor+for+real-time+monitoring+polynucleotide+kinase+activity.">A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.</a> Analytical Chemistry. 86 (16), 8481-8488 (2014).</li><li>Jiang, C., Yan, C., Jiang, J., Yu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+assay+for+T4+polynucleotide+kinase+activity+based+on+the+horseradish+peroxidase-mimicking+DNAzyme+combined+with+lambda+exonuclease+cleavage.">Colorimetric assay for T4 polynucleotide kinase activity based on the horseradish peroxidase-mimicking DNAzyme combined with lambda exonuclease cleavage.</a> Analytica Chimica Acta. 766, 88-93 (2013).</li><li>Castle, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Las1+interacts+with+Grc3+polynucleotide+kinase+and+is+required+for+ribosome+synthesis+in+Saccharomyces+cerevisiae.">Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae.</a> Nucleic Acids Research. 41 (2), 1135-1150 (2013).</li><li>Pillon, M. C., Sobhany, M., Stanley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+molecular+crosstalk+within+the+essential+Grc3/Las1+pre-rRNA+processing+complex.">Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex.</a> RNA. 24 (5), 721-738 (2018).</li><li>Geerlings, T. H., Vos, J. C., Raue, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+final+step+in+the+formation+of+25S+rRNA+in+Saccharomyces+cerevisiae+is+performed+by+5'--&gt;3'+exonucleases.">The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'--&gt;3' exonucleases.</a> RNA. 6 (12), 1698-1703 (2000).</li><li>Pillon, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+reveals+active+site+coordination+within+a+multienzyme+pre-rRNA+processing+complex.">Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex.</a> Nature Structural Molecular Biology. 26 (9), 830-839 (2019).</li><li>Solomatin, S., Herschlag, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+site-specific+labeling+of+RNA+with+fluorescent+dyes.">Methods of site-specific labeling of RNA with fluorescent dyes.</a> Methods in Enzymology. 469, 47-68 (2009).</li><li>Giusti, W. 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Described is an assay to measure kinase activity of Grc3 PNK on fluorescently-labeled nucleotide substrates. This protocol can be applied to characterize other PNK enzymes by adapting the Reaction Buffer and oligonucleotide substrate. For instance, the protocol calls for a trace amount of EDTA. The addition of EDTA is beneficial for two reasons: First, this approach favors magnesium-bound Grc3 by preventing the enzyme from binding to trace amounts of contaminating metals in the mixture. Second, a small amount of EDTA inh...

Polynucleotide kinases (PNK) play critical roles in many DNA and RNA processing pathways, such as DNA repair and ribosome assembly1,2,3,4,5. These fundamental enzymes catalyze the transfer of the terminal (gamma) monophosphate from a nucleoside triphosphate (NTP, most often ATP) to the 5&#39; hydroxyl end of a nucleotide substrate. One of the most well characterized PNKs is bacteriophage T4 PNK, which has broad substrate specificity and is heavily utilized by molecular biology labs for incorporating radioactive isotope labels onto the 5 terminus of a DNA or RNA substrate6,7,8,9,10,11,12. Another example of a PNK enzyme is CLP1, which is found in Eukarya, Eubacteria, and Archaea, and is implicated in several RNA processing pathways4,13,14,15.
Historically, most assays that measure polynucleotide kinase activity are dependent upon radioactive isotope labeling and subsequent autoradiography5,16. In recent years a number of additional assays have been developed to measure PNK activity, including single molecule approaches, microchip electrophoresis, molecular beacons, as well as colorimetric and luminescence-based assays17,18,19,20,21,22. While many of these new approaches provide enhanced detection limits and avoid the use of radioactivity, each has drawbacks, such as cost, reliance on immobilized resin, and limitations in substrate choice.
Grc3 is a polynucleotide kinase that plays a pivotal role in the processing of pre-ribosomal RNA2,3,23. Grc3 forms an obligate complex with the endoribonuclease Las1, which cleaves the Internal Transcribed Spacer 2 (ITS2) of the pre-ribosomal RNA3. Cleavage of the ITS2 by Las1 generates a product harboring a 5&#39; hydroxyl that is subsequently phosphorylated by the Grc3 kinase3. To investigate the nucleotide and substrate specificity of Grc3, an inexpensive assay that allowed testing of different oligonucleotide substrates was required. Therefore, a PNK phosphorylation assay using fluorescently-labeled substrates was developed. This assay was successfully used to determine that Grc3 can utilize any NTP for phosphoryl transfer activity, but favors ATP24. This protocol adapts the original assay to measure PNK activity of Grc3 on an RNA mimic of its pre-ribosomal RNA substrate (SC-ITS2, Table 1). One challenging aspect of this fluorescence-based approach is the reliance on large polyacrylamide gels to effectively resolve phosphorylated and nonphosphorylated substrates. The protocol provides specific details on how to pour these large gels and avoid common pitfalls when doing so.
Working with RNA requires particular care because it is strongly susceptible to degradation. There are simple preventative steps one can take to limit ribonuclease contamination. A separate RNA workstation that can be easily treated with an RNase inhibitor-containing cleaning agent is often helpful. Always wearing gloves when handling samples and use of RNase-free certified consumables is necessary. Because water is another common source of contamination, it is best to use freshly purified water and sterilize all solutions using a 0.22 &#956;m filter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm 34-well comb</td>
			<td>BioRad</td>
			<td>1653848</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 mm spacer</td>
			<td>BioRad</td>
			<td>1653812</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA ph 8.0</td>
			<td>KD Medical</td>
			<td>RGF-3130</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Magnesium Chloride</td>
			<td>KD Medical</td>
			<td>CAC-5290</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris pH 8.0</td>
			<td>KD Medical</td>
			<td>RGF-3360</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/Bis Solution 29:1</td>
			<td>BioRad</td>
			<td>1610146</td>
			<td></td>
		</tr>
		<tr>
			<td>5M Sodium Chloride</td>
			<td>KD Medical</td>
			<td>RGF-3720</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium persulfate (APS)</td>
			<td>BioRad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2383-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>Sigma</td>
			<td>B0394</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue sodium salt</td>
			<td>Sigma</td>
			<td>B5525-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Thomas Scientific</td>
			<td>1188K51</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoefer SQ3 Sequencer</td>
			<td>Hoefer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td><a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a></td>
		</tr>
		<tr>
			<td>Labeled RNA oligonucleotides</td>
			<td>IDT</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacia EPS 3500 Power Supply</td>
			<td>Pharmacia</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip 0. 22 um Filter</td>
			<td>Millipore</td>
			<td>5FCP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>BioRad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>tris base</td>
			<td>Sigma</td>
			<td>TRIS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 9500 gel imager</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure DEPC Water</td>
			<td>Invitrogen</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Glycerol</td>
			<td>Invitrogen</td>
			<td>19E1056865</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Fisher Chemical</td>
			<td>U15-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

nonradioactive assay to measure polynucleotide phosphorylation of small nucleotide substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

A successful representative denaturing gel of a titration of ATP with a fixed amount of Las1-Grc3 complex is shown in Figure 1. Addition of enzyme resulted in Las1-mediated RNA cleavage of the SC-ITS2 RNA substrate, leading to a defined RNA fragment (5-OH C2 RNA). Upon the addition of ATP, the C2 RNA fragment was phosphorylated by Grc3 PNK (5-P C2 RNA). In denaturing gels the phosphorylated RNA migrates faster than its unphosphorylated counterpart. As shown i...

Watch this Scientific Journal Video about Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates at JoVE.com

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

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