Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe

Xu, Man; Li, Baoxin

doi:10.1016/j.saa.2015.06.052

Cited by 20 publications

(5 citation statements)

References 23 publications

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“…The LFB showed good linear correlation to the concentration of NAD + from 0.5 to 1500 nM with a detection limit of 100 pM. The label-free detection of important analytes such as copper(II), 40 mutant DNA, 41 exonuclease activity, 42 streptavidin, 43 bisphenol A 44 with excellent performance have also been demonstrated as successful cases of SG I-based LFBs.…”

Section: Lfbs Based On Dsdna-binding Dyesmentioning

confidence: 90%

Label-free DNA-based biosensors using structure-selective light-up dyes

Guo

Hong

et al. 2016

Analyst

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Label-free biosensors (LFBs) have demonstrated a great potential in cost-effective applications, and most of the DNA-based LFBs are based on the principle of binding-induced structural transformation. This review is a collection of the latest reported studies, which have employed structure-selective nucleic acid dyes for the development of DNA-based LFBs. The collections in this review have been structured based on the selective binding of dyes towards specific DNA conformations, including single-stranded DNA, double-stranded DNA, triplex DNA, i-motifs and G-quadruplexes. The newest studies of employing versatile nucleases, fascinating nanomaterials, logic gates and cascades, DNA junctions and nanostructures have also been collected as examples. It is predicted that the imperative requirement for ultrahigh sensitivity, intelligent analysis, reliable detection, portable and fast assay would force the LFB development in a stimulative and continuous way.

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Section: Lfbs Based On Dsdna-binding Dyesmentioning

confidence: 90%

Label-free DNA-based biosensors using structure-selective light-up dyes

Guo

Hong

et al. 2016

Analyst

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“…The experimental setup was designed based on a previously published protocol to determine the activity of exonucleases [42]. Exonuclease III (Thermo Scientific) was chosen as a standard nuclease for this study at a concentration of 50U/ml.…”

Section: Exonuclease Assaymentioning

confidence: 99%

In silicoandin vitrocharacterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair

Baral

Bhattacharje

Dash

et al. 2023

Preprint

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Non–homologous end–joining DNA repair is essential for the survival and sustenance ofM·tuberculosis(Mtb) in the dormant stage of its life cycle. The ability of Mtb to sustain itself in the inactive form has been reported to be the critical factor for its resilience over the years. To unravel one of the salient features of the Mtb′s arsenal, we exploitedin silicoandin vitrotools to characterize the DNA binding properties of mycobacterial protein Ku (mKu) and its role in mycobacterial NHEJ. Here, we report the strong affinity of mKu for linear dsDNA exhibiting positive cooperativity for dsDNAs (40bp long or more). Molecular dynamics complemented within vitroexperiments showed that the DNA binding of mKu provides stability to both mKu homodimer and the DNA. Furthermore, mKu end capping of DNA was seen to protect the DNA termini against nucleolytic degradation by exonuclease. The DNA–mKu association formed higher–order oligomers probably due to the lodgement of two DNA molecules at opposite ends of the mKu homodimer. The ability of mKu to form continuous filament–like structures with DNA indicated its potential role in mycobacterial NHEJ synapsis.

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“…Although both nanomaterialbased methods are highly sensitive, they are not suitable for rapid diagnosis due to the complexity and time-consuming nature of nanomaterial synthesis, as well as the requirement of a skilled workforce [13][14][15][16][17]. Furthermore, methods utilizing fluorescent signals to detect Exo III activity, while convenient, exhibit suboptimal sensitivity [18]. However, there is still potential for further improvement in their analytical performance.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3′–5′ Exonuclease Assay

Jeung,

Han,

Lee

et al. 2023

Biosensors

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This study presents a technique for detecting 3′–5′ exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3′–5′ exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5′–end of a hairpin probe (HP), forming a blunt end. When the 3′–end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3′–end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

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Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe

Cited by 20 publications

References 23 publications

Label-free DNA-based biosensors using structure-selective light-up dyes

Label-free DNA-based biosensors using structure-selective light-up dyes

In silicoandin vitrocharacterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3′–5′ Exonuclease Assay

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