Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNK) play critical roles in many DNA and RNA processing pathways, such as DNA repair and ribosome assembly^1,2,3,4,5. These fundamental enzymes catalyze the transfer of the terminal (gamma) monophosphate from a nucleoside triphosphate (NTP, most often ATP) to the 5' 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 substrate^{6,7,8,9,10,11,12}. Another example of a PNK enzyme is CLP1, which is found in Eukarya, Eubacteria, and Archaea, and is implicated in several RNA processing pathways^4,13,14,15.

Historically, most assays that measure polynucleotide kinase activity are dependent upon radioactive isotope labeling and subsequent autoradiography^5,16. 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 assays^{17,18,19,20,21,22}. 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 RNA^2,3,23. 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' 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 μm filter.