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Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites.

ABSTRACT: DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X' and Y') for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X' and Y' to generate the X'Y' products. The resultant X'Y' products may in turn act as the templates to ligate probes X and Y for the generation of XY products, consequently inducing tricyclic LCR amplification under thermal denaturation conditions to generate a large number of XY products. The subsequent hybridization of XY products with the capture and reporter probes results in the formation of sandwich hybrids which may assemble on the 605QD surface to obtain 605QD-oligonucleotide-Cy5 nanostructures, inducing efficient FRET from the 605QD to Cy5 and the emission of Cy5. This nanosensor can detect DNA methylation at single 5-methylcytosine (5-mC) resolution with a detection limit of as low as 1.0 aM and a large dynamic range of 7 orders of magnitude. Moreover, this nanosensor can distinguish as low as a 0.01% methylation level, and it can detect DNA methylation in human lung cancer cells as well, holding great potential for accurate epigenetic evaluation and early cancer diagnosis.

SUBMITTER: Wang ZY

PROVIDER: S-EPMC5887231 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites.

Wang Zi-Yue ZY Wang Li-Juan LJ Zhang Qianyi Q Tang Bo B Zhang Chun-Yang CY

Chemical science 20171213 5

DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X' and Y') for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methyla ...[more]

PMID: 29675180

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Project description:Base excision repair (BER) is an important DNA repair pathway involved in the maintenance of genome stability. As the initiator of BER, DNA glycosylase can remove a damaged base from DNA through cleaving the N-glycosidic bond between the sugar moiety and the damaged base. Accurate quantification of DNA glycosylase is essential for the early diagnosis of various human diseases. However, conventional methods for DNA glycosylase assay usually suffer from poor sensitivity and complex probe design. Herein, we develop a single quantum dot-based nanosensor with multilayer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase (hAAG) using apurinic/apyrimidinic endonuclease 1 (APE1)-assisted cyclic cleavage-mediated signal amplification in combination with the DNA polymerase-assisted multiple cyanine 5 (Cy5)-mediated fluorescence resonance energy transfer (FRET). The presence of hAAG induces the cleavage of the hairpin substrate, generating a trigger. The resultant trigger can hybridize with a probe modified with an AP site, initiating the APE1-mediated cyclic cleavage to produce a large number of primers. The primers can subsequently initiate the polymerase-mediated signal amplification with a biotin-modified capture probe as the template, generating the biotin-/multiple Cy5-labeled double-stranded DNAs (dsDNAs). The resultant dsDNAs can assemble onto the QD surface to form the QD-dsDNA-Cy5 nanostructure, leading to efficient FRET from the QD to Cy5 under the excitation of 405 nm. In contrast to the typical QD-based FRET approaches, the assembly of multilayer of multiple Cy5 molecules onto a single QD significantly amplifies the FRET signal. We further verify the FRET model with one donor and multilayered acceptors theoretically and experimentally. This single QD-based nanosensor can sensitively detect hAAG with a detection limit of as low as 4.42 × 10-12 U ?L-1. Moreover, it can detect hAAG even in a single cancer cell, and distinguish the cancer cells from the normal cells. Importantly, this single QD-based nanosensor can be used for the kinetic study and inhibition assay, and it may become a universal platform for the detection of other DNA repair enzymes by designing appropriate DNA substrates.

Dataset Information

Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites.

Publications

Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites.

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