Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

Abstract

As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5'-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.

Hierarchical Metal–Organic Framework-Confined CsPbBr3 Quantum Dots and Aminated Carbon Dots: A New Self-Sustaining Suprastructure for Electrochemiluminescence Bioanalysis

“…Abnormal T4 PNK activity has been closely associated with several human genetic syndromes, so screening of its inhibitors is of great importance in drug development and clinical treatment. − In this work, (NH 4 ) 2 SO 4 and ADP are selected as model inhibitors. (NH 4 ) 2 SO 4 can inactivate T4 PNK by changing its configuration, and ADP can interfere with the phosphate group transfer process from ATP to DNA.…”

“…Accordingly, excellent ECL performance in terms of stability and efficiency is achieved from the PeQDs-NCDs@HZIF-8. To illustrate the potential application of PeQDs, the ternary nanocomposite is then employed for ECL evaluation of T4 polynucleotide kinase (T4 PNK) activity, which plays a crucial role in DNA repair and cellular metabolism. − In this strategy (Scheme ), we design a multifunctional DNA probe with a 5′-hydroxyl terminal, which undergoes phosphorylation of T4 PNK and cleavage of λ-exonuclease (λ-Exo) to activate the fragments for hybridization chain reaction (HCR) and electrode capture. Afterward, the HCR is triggered to generate a large amount of double-stranded DNA (dsDNA) for the assembly of rhodamine 6G (Rh6G).…”

Detection of dibutyl phthalate in food samples by fluorescence ratio immunosensor based on dual-emission carbon quantum dot labelled aptamers

“…In recent years, they have been widely used in several analytical techniques because of their high ability to bond specifically with target substances and their easy modification and fixation (Cruz-Aguado & Penner, 2008;Duan et al, 2012;Espiritu et al, 2018;Fenzl et al, 2017;Noonan et al, 2013). Aptamers are often used in combination with fluorescent labelling materials to achieve sensitive and intuitive detection (Gao et al, 2017;Wu et al, 2018). Currently, the most widely used fluorescent labelling materials are organic fluorescent dyes, fluorescent proteins, and quantum dot materials.…”

A novel fluorescent sensor based on 4-(diethylamino)-2-(hydroxy)-phenyl imine functionalized naphthalimide for highly selective and sensitive detection of CN– and Fe3+

“…[25][26][27] Compared with ion chromatography, 28 flow injection analysis 29 and titration analysis detection methods, 30 fluorescence method is simple, low cost and widely used. [31][32][33] In view of these and as part of our research interest in ions recognition, [34][35][36][37][38] herein, we reported a novel sensor molecule (HB1) based on 4-(diethylamino)-2-(hydroxy)-phenyl imine functionalized naphthalimide derivative (Scheme 1). Interestingly, the sensor molecule HB1 could not only fluorescence detection CNin DMSO/H 2 O (8 : 2, v/v) solution but also dual-channel (absorption and fluorescence) detection of Fe 3+ in DMSO solution with high selectivity and sensitivity.…”