Fluorescent probes for the analysis of DNA strand scission in base excision repair

Abstract

We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5′- and 3′-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using 32P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect the probes from nonspecific nucleases, and the base excision repair activity was successfully detected in HeLa cells.

A label-free and sensitive fluorescent method for the detection of uracil-DNA glycosylase activity

“…1D, the method was found to be capable of detecting UDG activity as low as 0.0008 U mL À1 based on 3s b /S, in a range of at least 0.0008-0.1 U mL À1 . This sensitivity was comparable or even better than those in the previously reported fluorescent methods for UDG activity assays, [13][14][15][16][17][18][19][20][21][25][26][27]34 most probably ascribed to its characteristics of one uracil removal in each DNA substrate without the need of DNA conformational changes. The results of polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis on the DNA duplex in the absence and presence of UDG clearly showed that the uracil in DNA-U was removed by UDG regardless of the presence of DNA-X or ATMND (Fig.…”

“…12 For instance, pairs of fluorophores and quenchers were attached at the ends of uracil-containing DNA substrates to measure UDG activity via fluorescence enhancement. [13][14][15] Nanomaterials including graphene oxide and gold nanoparticles were also utilized as quenchers and colorimetric reporters, respectively, for optical UDG assays. 16,17 Besides, the activation of uracil-containing DNAzymes by UDG was applied for more sensitive detection of UDG activity through DNAzyme-based signal amplifications.…”

“…Nevertheless, these label-free fluorescent approaches for UDG activity detection, 25,26 as well as many labelled probes reported before, [13][14][15][16][17][18] are based on simultaneous removal of multiple uracil bases to induce the conformational changes of DNA for signal transduction. Such DNA substrates containing multiple uracil sites are efficient but may make the estimation of UDG activity relatively complicated, because it can give little signal even if some (not all) of the uracil sites in each DNA substrate are removed but insufficient to induce the DNA conformational changes.…”

Inhibition of DNA Glycosylases via Small Molecule Purine Analogs

“…This basic strategy contrasts with the uracil-directed, tethered ligand strategy developed by the Stivers group for discovery of UDG2 inhibitors [34], or the hairpin strategy utilized by the Iwai group for probing cellular BER activity [35]. While these models were efficient in the identification of novel inhibitors [36], [37] and allowed for detection of glycosylase activity, neither approach was designed or adapted to probe large libraries of small molecules as part of a high-throughput screen.…”

NEIL1 Binding to DNA Containing 2′‐Fluorothymidine Glycol Stereoisomers and the Effect of Editing

“…We then protected both hydroxyl groups of the thymine glycol with acetyl groups. [20,21] The resulting compounds 3a, 3b were treated with trifluoroacetic acid to give the 5,6- O -acetyl-2′-ribofluorothymidine glycols 4a, 4b which were converted to the phosphoramidite building blocks 6a, 6b by standard procedures.…”