Unimolecular Chemically Modified DNA Fluorescent Probe for One-Step Quantitative Measurement of the Activity of Human Apurinic/Apyrimidinic Endonuclease 1 in Biological Samples

Abstract

A novel DNA structure containing a 3' internal-loop modified abasic site has been constructed which enables effective differentiation between apurinic/apyrimidinic endonuclease (APE1) and nonspecific endonuclease (DNase I). When this unique substrate structure is employed, a double-loop frayed-end chimeric fluorescent probe is successfully developed for quantitative measurement of the activity of APE1 in biological samples without the need of additional cleanup or preconcentration steps. The method is simple and rapid and has a single-step with a linear working range between 0.1 and 5.0 U/mL and a lower limit of detection of 0.1 U/mL. It holds great potential in real-time monitoring of the variation of intracellular and extracellular APE1, which will be very useful for further understanding of the DNA repair pathways in different organisms.

A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells

“…This is more sensitive than the previously developed DNA fluorescent probe for in-vitro APE1 detection (13). Figure 3C summarizes the selectivity of Nanoprobe A for APE1 against other possibly coexisting nucleases.…”

“…Abnormal expression/localization of APE1 has been found in tumor cells (6–8). Up to now, a number of approaches have been developed for the detection of APE1, such as enzyme-linked immunosorbent assay (9), electrochemical immunoassay (10), electrochemiluminescence immunosensor (11) and fluorescent DNA probes (12,13). However, most of them are only suitable for in-vitro applications.…”

Fluorescent Probes of DNA Repair

“…Additionally, the probes could be used to measure enzyme inhibition. Following the work of Saparbaev, others have reported similar hUNG and APE1 probes that provide some improvements to the original probe design such as eliminating the need for multiple uracil residues. − A similar probe of endonuclease III-like protein 1 (NTH1) was reported, which replaces uracil with damaged thymine bases, and a molecular beacon based probe of phosphodiesterase 1 has also been reported . A strand displacement assay of DNA polymerases reported by the group of Simeonov was later used by Coggins and co-workers to screen small molecule inhibitors and perform a SAR study on several hits .…”

Metal-Ion-Responsive Bionanocomposite for Selective and Reversible Enzyme Inhibition

“…Ten mM Tris buffer (pH 8.0) could only release about 12% of the APE1 bound on MIP-2. Since 1× Buffer 1.1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl 2 , 100 μg/mL BSA, pH 7.0) is the most commonly used buffer for the AP-endonucleolytic reactions of APE1, we tested the use of 5× or 10× Buffer 1.1 to release the APE1 bound on MIP-2 according to our previous work. , As shown in Figure A, 5× Buffer 1.1 released about 80% of the bound APE1, and the dissociation efficiency of 10× Buffer 1.1 was close to 100%. Different from the results of MIP-2, 10× Buffer 1.1 could only release about 50% of the bound APE1 from SiMNP@AVD-2, suggesting different interactions between APE1 and the two different NPs (Figure S6).…”