Homogeneously Sensitive Detection of Multiple DNA Glycosylases with Intrinsically Fluorescent Nucleotides

Abstract

DNA glycosylases are responsible for recognition and excision of the damaged bases in the base excision repair pathway, and all mammals express multiple DNA glycosylases to maintain genome stability. However, simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a rapid and sensitive fluorescent method for simultaneous detection of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycolase (UDG) using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG. In addition, we design 2-AP and P-dC signal probes as the hairpin structures with 2-AP and P-dC in the stems. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals, with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG. This method can simultaneously detect multiple DNA glycosylases with the detection limits of 0.0035 U/mL for hOGG1 and 0.0025 U/mL for UDG, and it can even measure DNA glycosylases at the single-cell level. Moreover, this method can be applied for the measurement of enzyme kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential for further applications in biomedical research and clinical diagnosis.

Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background

“…13,[16][17][18]22 Moreover, the detection limit of the proposed method is directly measured to be 1 cancer cell, which is much higher that of the singlemolecule counting-based uorescence assay (9 cells), 21 single QD-based uorescent nanosensor (5 cells), 20 and l exo-assisted recycling amplication-based uorescence assay (3 cells). 23 These results clearly demonstrate that the proposed method can be applied for accurate detection of hOGG1 activity in crude cell extracts with high sensitivity.…”

“…The resultant products can be label-free and real-time monitored with SYBR Green I (a uorescent dye for selectively staining dsDNA) as the indicator for quantifying DNA glycosylase activity. In comparison with the reported DNA glycosylase assays, 13,[16][17][18][20][21][22][23] this strategy has signicant advantages: (1) this assay enables the real-time monitoring of DNA glycosylase activity with a wide dynamic range, (2) the selfdirected autocycling polymerization and SDS can result in the exponential DNA amplication, (3) the four primers can recognize multiple distinct regions of the DNA template to inhibit the nonspecic amplication, achieving the zero background, (4) the assay reaction is implemented in one tube with involvement of only a single type of polymerase under isothermal conditions, and the output signal can be detected in a label-free manner, greatly reducing the assay costs.…”

“…The sensitivity of the proposed method has been improved by 4 orders of magnitude compared with that of the colorimetric assay based on DNA-AuNP probes (7.0 Â 10 À4 U mL À1 ), 18 220.0-fold compared with that of singlemolecule counting-based uorescence assay (2.2 Â 10 À6 U mL À1 ), 21 180.0-fold compared with that of a single QD-based uorescent nanosensor (1.8 Â 10 À6 U mL À1 ), 20 and is comparable to those of exonuclease (i.e., Exo III and l exo)-directed cycling signal amplication-based uorescence assays (1.0 Â 10 À9 and 3.0 Â 10 À9 U mL À1 ). 22,23 The improved sensitivity can be attributed to the high efficiency of DNA repairing-driven selfdirected exponential replication and the zero background signal resulting from the efficient inhibition of nonspecic amplication by the high specicity of multiple primer-dependent amplication.…”

“…Besides their laborious procedures, HPLC and MS usually suffer from high background signals due to the generation of articial damaged bases during sample collection and preparation, while ELISA may underestimate the actual damaged base level owing to the loss of samples during multi-step washing. The latter mode can overcome these limitations by directly detecting DNA products with AP sites, driving the development of colorimetric, 18 electrochemical, 19 and uorescent [20][21][22][23][24] methods. Despite their improved performances, each method has its own limitations for practical applications.…”

“…19 The uorescence assays based on quantum dot (QD) nanosensor and DNA repair-responsive molecular beacons enable effective detection of DNA glycosylase activities, 20,21 but they involve complicated manipulations. The introduction of enzymes such as exonuclease III (Exo III), 22 lambda exonuclease (l exo) 23 and endonuclease IV (Endo IV) 24 for target signal amplication has greatly improved the detection sensitivity, but they suffer from high cost for the preparation of uorophoreand quencher-labeled probes, and low specicity and high background caused by the nonspecic nuclease digestion. Therefore, the development of simple, cost-effective, specic and sensitive methods for the DNA glycosylase assay is highly desirable.…”