Detection of T4 polynucleotide kinase activity based on cationic conjugated polymer-mediated fluorescence resonance energy transfer

Abstract

A simple but robust strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity is developed by means of a DNA phosphorylation-accelerated λ exonuclease cleavage reaction coupled with cationic conjugated polymer (CCP)-mediated fluorescence resonance energy transfer (FRET). Firstly, a label-free hairpin DNA with a 5′-hydroxyl end is designed as the substrate of T4 PNK. SYBR Green I (SGI), a double-stranded DNA (dsDNA)-specific fluorescent dye, can fluoresce only when intercalated to the stem region of the hairpin DNA. When mixed with CCP, the SGI-binding hairpin DNA will be brought in close proximity with the CCP due to strong electrostatic interaction, leading to efficient FRET from CCP to SGI. However, in the presence of T4 PNK, the hairpin DNA would be phosphorylated at its 5′-terminus and thus can be immediately recognized as the initial cleavage site of λ exonuclease. The phosphorylation-actuated λ exonuclease reaction will cleave the stem of the hairpin to yield a single-stranded DNA, which is unable to combine with SGI and as a result, the FRET signal would decrease gradually in correlation to the T4 PNK activity. Therefore, by recording the change of FRET ratio, T4 PNK activity can be facilely determined in a mix-and-read manner. Due to the light harvesting and fluorescence amplification properties of CCP, high sensitivity is achieved for this homogeneous assay. This new strategy provides a simple detection procedure, easy readout and cost-effective manner for T4 PNK analysis, which shows great potential in the study of polynucleotide kinase-related biological processes.