A hairpin-type DNA probe for direct colorimetric detection of endonuclease activity and inhibition based on the deaggregation of gold nanoparticles
- 107 Downloads
A method is described for the determination of the activity of endonuclease. It based on the deaggregation of gold nanoparticles (AuNPs) aggregated by the action of poly(diallyldimethylammonium chloride) (PDDA). A single-stranded DNA (ssDNA) is released after enzymatic cleavage catalyzed by endonuclease. The released fragments bind electrostatically to PDDA and inhibit the PDDA-induced aggregation of AuNPs. This is accompanied by a color change from blue to red and a decrease in the absorption ratio (A630/A520). Under the optimal conditions, this ratio increases linearly in the 0.001 to 1 U·μL−1 EcoRI endonuclease activity range. The detection limit is of 2 × 10−4 U·μL−1 which is much better or at least comparable to previous reports. The method is deemed to have wide scope in that it may be used to study other endonuclease activity (such as BamHI) by simply changing the specific recognition site of the hairpin-like DNA probe. The assay may also be employed to screening for inhibitors of EcoRI endonuclease.
KeywordsEcoRI Gold nanoparticle Visual test Deaggregation Endonuclease inhibitor
This work was funded by the Natural Science Foundation of China (NSFC) (No. 21407035), Shandong Provincial Natural Science Foundation (ZR2014BM021), Technology and Development Program of Weihai (2014DXGJ15), HIT-NSRIF (2011101).
Compliance with ethical standards
All procedures were in compliance with ethical strandards.
- 7.Alves J, Rulter T, Geiger R, Fliess A, Maass G, Pingoud A (1989) Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymic activity, not, however, the preference of this restriction endonuclease for cleavage within the site -GAATTC. Biochemistry 28:2678–2684CrossRefGoogle Scholar