Nanofluidic sensing platform for PNK assay using nonlinear hybridization chain reaction and its application in DNA logic circuit.

In this study, a polyethyleneimine (PEI)/Zr4+-functionalized nanofluidic sensing platform based on nonlinear hybridization chain reaction (NHCR) was developed for PNK activity assay. With the existence of PNK, the hairpin HPNK was cleaved by λ exonuclease, liberating the initiator T-DNA. Then T-DNA triggered the nonlinear HCR in solution and the reaction products were absorbed onto the nanopore, which changed the surface charge of nanofluidic device and could be detected by current-voltage characteristic curves. Compared to traditional linear HCR, the nonlinear HCR exhibits a higher sensitivity and order of growth kinetics, making it a powerful signal amplifier in bioanalysis. Due to the powerful amplification efficiency of nonlinear HCR, high sensitivity of the nanopore and specific recognition site of PNK/λ-Exo, an ultrasensitive and selective PNK sensing approach had been developed and applied to precisely quantitate the PNK activity with a LOD of 0.0001 U/mL. Moreover, utilizing this nanofluidic system as a foundation, we constructed a logic circuit that utilized PNK, adenosine diphosphate (ADP), and (NH4)2SO4 as input elements. ADP and (NH4)2SO4 had a crucial function in facilitating the PNK to regulate the DNA logic gate. By modifying the target and inhibitors, the nanofluidic device could detect a variety of stimuli and execute more advanced logical operations.

Electrochemical DNA Sensors Based on MoS2-AuNPs for Polynucleotide Kinase Activity and Inhibition Assay.

The determination of T4 polynucleotide kinase (PNK) activity and the screening of PNK inhibitors are critical to disease diagnosis and drug discovery. Numerous electrochemical strategies have been developed for the sensitive measurement of PNK activity and inhibition. However, they often suffer from additional labels and multiple steps of the detection process for the electrochemical readout. Herein, we have demonstrated an electrochemical DNA (E-DNA) sensor for the one-step detection of PNK with "signal-on" readout with no need for additional labels. In our design, the highly switchable double-stranded DNA (dsDNA) probes are immobilized on the gold nanoparticle-decorated molybdenum disulfide nanomaterial (MoS2-AuNPs), which possesses large surface area and high conductivity for elevating the signal gain in the PNK detection. This signal-on E-DNA sensor integrated with MoS2-AuNPs exhibits a much higher sensitivity than that without MoS2-AuNPs, showing a detection limit of 2.18 × 10-4 U/mL. Furthermore, this assay shows high selectivity, with the ability to discriminate PNK from other enzymes and proteins, and can be utilized to screen inhibitors. The proposed sensor is easy to operate with one-step readout and robust for PNK detection in the biological matrix and shows great potential for point-of-care in clinical diagnostics and drug screening.

A novel microchip electrophoresis laser induced fluorescence detection method for the assay of T4 polynucleotide kinase activity and inhibitors.

T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5'-hydroxyl termini in nucleic acids, which play a crucial role in DNA recombination, replication and damage repair. Here, a microchip electrophoresis laser induced fluorescence (MCE-LIF) method based on biochemical reaction was developed for the detection of T4 PNK activity and inhibitors. In this method, the single strand DNA (ssDNA) was hybridized with the 5-carboxyfluorescein (FAM) labeled single strand DNA (ssDNA-FAM) to form FAM labeled double-stranded DNA (dsDNA-FAM). In the presence of T4 PNK and adenosine triphosphate (ATP), T4 PNK catalyzes the transfer of γ-phosphate residues from ATP to the 5-hydroxyl terminal of dsDNA-FAM. The phosphorylated dsDNA-FAM can be gradually hydrolyzed by λexo to produce a FAM labeled single nucleotide fragment. Then the FAM labeled single nucleotide fragment and the unhydrolyzed dsDNA-FAM were separated by MCE, and two electrophoresis peaks appeared in the electrophoretogram. The detection of T4 PNK activity and inhibitors was realized by measuring the peak height of the FAM labeled single nucleotide fragment in electrophoretogram. This assay is very sensitive with a limit of detection of 0.002 U/mL, and it can be further used to screen the T4 PNK inhibitors.

An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification.

Polynucleotide kinase (PNK) plays an essential role in cellular nucleic acid metabolism and the cellular response to DNA damage. However, conventional methods for PNK assay suffer from low sensitivity and involve multiple steps. Herein, we develop a simply electrochemical method for sensitive detection of PNK activity on the basis of Au nanoparticle (AuNP)-mediated lambda exonuclease cleavage-induced signal amplification. We use [Ru(NH3)6]3+ as the electrochemically active indicator and design two DNA strands (i.e., strand 1 and strand 2) to sense PNK. The assembly of strand 2 on the AuNP surface leads to the formation of AuNP-strand 2 conjugates which can be subsequently immobilized on the gold electrode through the hybridization of strand 1 with strand 2 for the generation of a high electrochemical signal. The presence of PNK induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH3)6]3+ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH3)6]3+ peak current signal. This assay is very sensitive with a detection limit of as low as 7.762 × 10-4UmL-1 and exhibits a large dynamic range from 0.001 to 10UmL-1. Moreover, this method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis.

Quencher-free hairpin probes for real-time detection of T4 polynucleotide kinase activity.

Traditional methods of assaying polynucleotide kinase (PNK) activity are discontinuous, time-consuming, and laborious. Here we report a new quencher-free approach to real-time monitoring of PNK activity using a 2-aminopurine probe. When the 2-aminopurine probe was 5'-phosphorylated by PNK, it could be efficiently degraded by lambda exonuclease to release free 2-aminopurine molecules and generate a fluorescence signal. This method not only provides a universal approach to real-time monitoring of PNK activity, but also shows great potential for screening suitable inhibitor drugs for PNK.

Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification.

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 5' to 3' direction by lambda exonuclease (λ exo) when its 5' end is phosphorylated by PNK. So, the 3' stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0×10(-4)U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases.