Homo-FRET enhanced ratiometric fluorescence strategy for exonuclease III activity detection

Zhang, Xu; Bai, Yan; Jiang, Yongjian; Wang, Na; Yang, Feifan; Zhan, Lei; Huang, Chengzhi

doi:10.1039/d0ay02315a

Cited by 6 publications

(3 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Exonuclease III ( Exo III) has a high exodeoxyribonuclease activity to produce stretches of single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA) in its 3′ to 5′ direction. , Different from the traditional target gene amplification strategy, Exo III-assisted amplification performs target DNA detection by direct amplification of signaling reagents. , In addition, Exo III-assisted target recycling amplification is usually employed to amplify signal strength for in vitro diagnosis of nanomaterials but rarely used for direct detection of pathogenic nucleic acids from clinical samples. − …”

Section: Introductionmentioning

confidence: 99%

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids

Zhou,

Zhang,

Sun

et al. 2023

ACS Synth. Biol.

View full text Add to dashboard Cite

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/μL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.

show abstract

Section: Introductionmentioning

confidence: 99%

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids

Zhou,

Zhang,

Sun

et al. 2023

ACS Synth. Biol.

View full text Add to dashboard Cite

show abstract

“…However, despite their widespread use, both methods are time-consuming, labor intensive, and pose safety risks during the detection process [9,10]. Recently, alternative methods for detecting 3 -5 exonuclease activity have been developed, such as fluorescence-based methods utilizing the G-quadruplex [11][12][13][14][15] and FRET [16][17][18][19]. In addition, a detection method using metal nanoparticles, including copper nanoparticles [20,21], has been developed for detecting 3 -5 exonuclease activity.…”

Section: Introductionmentioning

confidence: 99%

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity

Han

Jeung

Jang

et al. 2023

IJMS

View full text Add to dashboard Cite

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3′-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria’s activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

show abstract

“…Exonuclease III (Exo III), which belongs to the exonuclease family, exhibits 3 -5 exonuclease activity. It hydrolyzes the blunt 3 -end of dsDNA without recognizing a specific site but exhibits low activity against ssDNAs and dsDNAs with a 3 overhang [2,3]. Previous studies have shown that 3 -5 exonucleases perform several crucial roles in cellular and physiological processes [4].…”

Section: Introductionmentioning

confidence: 99%

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

Jeung,

Han,

Lee

et al. 2023

Biosensors

View full text Add to dashboard Cite

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.

show abstract

Homo-FRET enhanced ratiometric fluorescence strategy for exonuclease III activity detection

Abstract: In this work, homo-FRET (Förster resonance energy transfer between the same kind of fluorophores) taken place in a hetero-FRET (FRET between two different fluorophores) systems can effectively improve the energy...

Cited by 6 publications

References 27 publications

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity

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

Contact Info

Product

Resources

About