Uracil DNA glycosylase UDG is a

Uracil-DNA glycosylase (UDG) is a highly conserved damage repair enzyme which can specifically recognize and excise uracil residue within the DNA sequences and actuate the DNA Trehalose australia excision repair (BER) pathway which keeps the maintenance of genomic integrity and stability [[21], [22], [23]]. However, the abnormal UDG activity in human cells may cause malfunction of the BER pathway and eventually various diseases such as human immunodeficiency, lymphoma, bloom syndrome, neurodegeneration, and cancers [22,[24], [25], [26], [27]]. Hence, the accurate and sensitive assay of UDG activity is of great importance for biomedical research and clinical diagnosis. Herein, we construct a facile and robust SERS platform for highly sensitive and reproducible detection of UDG using target-activated plasmonic coupling. This SERS system consists of DNA substrates and Raman dyes-modified gold nanoparticles (AuNPs) that is used for the specific recognition of target and SERS signal output, and two kinds of tool enzymes, exonuclease I (Exo I) and endonuclease IV (Endo IV) that is utilized to degrade DNA substrates on AuNPs for controllable self-assembly of plasmonic nanoparticles. On the basis of the ingenious design of this SERS system, self-assembled hot spots with narrow EFs and uniform interparticle spacing can be readily constructed by target-induced dual enzymatic cleavage reaction, allowing SERS detection of target UDG with high sensitivity and reproducibility. In addition, it is deserved to be mentioned that the target-mediated plasmonic coupling-based strategy can be easily extended for other damage repair enzymes detection (e.g. 8-oxoguanine glycosylase, hOGG1) by using corresponding DNA substrate. More importantly, simultaneous detection of multiple analytes can be achieved by the utilization of different substrates-decorated AuNPs coded with different Raman dyes [21,28]. As a result, the developed strategy is able to afford high throughput detection with improved operational convenience and technical robustness. Hence, this proposed strategy indeed creates a simple and universal SERS platform for highly sensitive detection of damage repair enzymes and related biomedicine research and clinical diagnosis.
Materials and methods
Results and discussion
Conclusions In summary, we construct a simple and robust SERS platform for highly sensitive and selective detection of UDG based on target-induced plasmonic coupling. The operation of SERS system, activated by target-activated dual enzymatic cleavage reaction, contributes to controllable self-assembly of plasmonic nanoparticles. Thus self-assembled hot spots with narrow EFs and uniform interparticle spacing can be readily generated, allowing homogenous SERS detection of UDG with high sensitivity and reproducibility. Compared with the existing methods for UDG detection, the present strategy achieves improved sensitivity with a detection limit as low as 4.29 × 10−4 U mL-1 and widened dynamic range from 1.0 × 10-3 U mL-1 to 10 U mL-1. Furthermore, this strategy can be easily extended for other damage repair enzymes detection through the design of corresponding DNA substrates. Importantly, simultaneously monitoring multiple analytes can be realized by using different Raman-dyes coded plasmonic nanoparticles. Therefore, the proposed strategy constructs a useful and practical platform for SERS sensing of damage repair enzyme with high sensitivity and superb selectivity. This platform also holds great potential to be applied for high throughput assays for drug screening and clinical diagnostics.
Acknowledgements This work was supported by National Natural Science Foundation of China (31471644), the Primary Research & Development Plan of Shandong Province (2017GSF220009), the program for Taishan Scholer of Shandong provinces (ts201712048) and the Natural Science Foundation of Shandong Province of China (ZR2016BL27).
Introduction DNA is vulnerable to damage from persistent endogenous and environmental stress [1], [2], and DNA damage may initiate carcinogenesis as a result of mutations and exacerbating replication errors [3], [4], [5]. Base excision repair (BER) may correct DNA damage from alkylation, deamination and oxidation [6] and its repair pathway is initiated by specific DNA glycosylase that catalyzes the cleavage of the N-glycosidic bond, liberating the damaged base and generating an abasic site (AP site) and ultimately coordinating with other repair proteins to accomplish the whole DNA repair [7]. The abnormal level of DNA glycosylase in human cells may cause the malfunction of base excision repair and eventually various diseases such as cancers [8] and neurological diseases [9], suggesting the great potential of DNA glycosylases in cancer diagnosis and treatment [10], [11].