Determination of Endonuclease Activity by an Enzyme-Free Fluorescent Biosensor Using the Hybridization Chain Reaction (HCR)

Abstract

A fluorescent labeling system for the determination of endonuclease activity using the hybridization chain reaction (HCR) is reported. Two hairpin probes, H1 and H2, were well-designed in which two split cleavage sequences of endonuclease EcoRI were respectively integrated into H1 and H2. Black Hole Quencher 1 (BHQ1) and carboxyfluorescein (FAM) were labeled at the 5′ and 3′ ends of H1, respectively. Once trigger DNA was introduced, HCR between H1 and H2 was triggered to form long DNA nanowires containing the intact cleavage sequences of EcoRI. At the same time, the BHQ1 and FAM were close together and the fluorescence signal was quenched. In the presence of EcoRI, the cleavage sequences were recognized by EcoRI and long DNA nanowires were cleaved to short double-stranded DNA (dsDNA) polymers. The FAM was far from BHQ1, resulting in the recovery of fluorescence signal. Under the optimized conditions, the fluorescence intensity was linear with EcoRI concentration over the range from 0.001 to 0.2 U μL⁻¹ and the detection limit is 1.2 × 10⁻⁴ U µL⁻¹. In addition, this strategy is versatile and may be used to determine other endonucleases activity by simply changing the specific cleavage sites in HCR system.

Keywords:

• Endonuclease activity
• endonuclease EcoRI
• fluorescence
• hybridization chain reaction (HCR)

Disclosure Statement

There are no conflicts of interest to declare.

Ethical approval of the use of human subjects

All experiments were performed in accordance with the guidelines (approval number 20170201) that was granted on 20 August 2019.

Additional information

Funding

This work was financially supported by the National Natural Science Foundation of China (grant number 21974091) and the Cooperative Project of Analysis and Testing Center of Sichuan Academy of Agricultural Science (grant numbers 20H1090 and 0020303410001).