A renewable DNA biosensor for sensitive detection of DNA methyltransferase activity based on cascade signal amplification
Introduction
DNA methylation is the most frequent and important epigenetic modification in mammalian genomic DNA [[1], [2], [3]]. In many human diseases, including cancers, abnormal DNA methylation levels have been found [4,5]. The modification process involves the transfer of methyl group from S-adenosylmethionine (SAM) to adenine or cytosine bases under methyltransferase (MTase) catalysis. As a potential biomarker, alterations in MTase activity will lead to a wide range of physiological or pathological changes [[6], [7], [8]]. Therefore, detection of MTase activity is of great significance and has attracted increasing attention [9].
Recently, many new methods for detecting MTase activity have been developed, such as fluorescence [[10], [11], [12]], electrochemical [[13], [14], [15], [16], [17]], electrochemiluminescence [18,19], and colorimetric [[20], [21], [22]] methods. Meanwhile, various amplification strategies are employed to amplify the signal for highly sensitive detection, including rolling circle amplification (RCA) [[23], [24], [25]], strand displacement amplification (SDA) [26,27], hybridized chain reaction (HCR) [[28], [29], [30]], and exonuclease assisted signal amplification [31,32]. Most of these methods require sophisticated DNA sequence design for efficient amplification and low background signal [33].
Invasive reaction is based on specific recognition of a single-base overlapping structure by flap endonuclease I (FENI) [34]. Since the cleavage reaction is based on the recognition of nucleic acid structure rather sequence, it can be used to construct a signal amplification reaction that do not require complex sequence designs [35]. In addition, by simply combining two invasive reactions in series, a 107-fold cascade signal amplification can be achieved. However, as far as we know, few methods for detecting DNA MTase activity assay based on invasion reactions have been reported.
Herein, we constructed a simple DNA biosensor based on cascaded invasive reactions for highly sensitive MTase assay. The hairpin DNA biosensor was first methylated by MTase and then cleaved into single-stranded DNA. The single-stranded DNA further triggered cascaded invasive reactions that produce amplified fluorescent signals. Furthermore, the DNA biosensor was immobilized on magnetic beads, and could be regenerated after detection by a simple DNA polymerase treatment. The renewable DNA biosensor enables highly sensitive, specific, and economical DNA methyltransferase detection and can be extended to apply to other enzymes assays.
Section snippets
Construction of the biosensor
The 5’-end biotinylated sensing probes were immobilized on the surface of Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (1 μm in diameter) according to the manufacturer's recommendations, and resuspended in 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at 4 °C. The amount of beads and probes was adjusted to 10 pmole of probes per milligram of beads. Sequence of sensing probe was shown in Table S1.
Cascade invasive reactions
Twenty microliters of a reaction mixture contained 10 mM MOPS (pH 7.5), 0.05% (v/v) NP-40,
Design of the DNA biosensor for Dam MTase assay
A scheme of the biosensor for Dam MTase assay is illustrated in Fig. 1A. The hairpin sensing probe was first immobilized on the surface of the magnetic bead. And a Cy3-modified DNA oligonucleotides which complementary to sensing probe was used to characterize the sensing probes-coated beads (Figs. S1 & S2). In the presence of Dam MTase, the adenines (A) in recognition site (5’- GATC-3' / 3’-CTAG-5’) on the probe are methylated. Then, the sensing probe was cleaved by DpnⅠ endonuclease at the
Conclusions
In summary, we have developed a biosensor for DNA methyltransferase activity assay based on cascade invasive reactions. Dam MTase was employed as model DNA methyltransferase. The methylation and cleavage of the sensing probe will trigger cascade signal amplification invasive reactions. The efficient signal amplification ensures the high sensitivity of our proposed biosensor. And the detection limit was 0.002 U/mL for Dam MTase activity assay. Because the sensing probe was fixed on the magnetic
CRediT authorship contribution statement
Yunlong Liu: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing, Funding acquisition. Yuanbiao Tu: Methodology, Investigation, Data curation. Haiping Wu: Validation, Formal analysis. Hang Zhang: Data curation, Validation. Honghong Chen: Data curation, Formal analysis. Guohua Zhou: Resources, Writing - review & editing. Peng Wang: Visualization, Writing - review & editing. Yueqing Gu: Supervision, Funding acquisition, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by National Natural Science Foundation of China (61901527, 81729002, 91859204 and 81727804), the Priority Academic Program Development of Jiangsu Higher Education and State key laboratory of Natural Medicines (SKLNMZZCX201819).
Yunlong Liu He obtained his Ph.D. degree in basic medicine from Nanjing University in 2017. Now he is a post-doctoral in China Pharmaceutical University. His current research interests are biosensor and molecular diagnosis.
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2022, Sensors and Actuators B: ChemicalCitation Excerpt :The proposed method was compared with other methods for the detection of DNA MTase activity in terms of analytical performance (Table 1). The LOD of the proposed method is almost at the lowest level compared with other reported methods based on single amplification [17,19,20,47,51] or cascade amplification [48,49,54] strategies. This could be attributed to the combined effect of SDA and MNAzyme cascade amplification system and AuNPs as element tags.
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Yunlong Liu He obtained his Ph.D. degree in basic medicine from Nanjing University in 2017. Now he is a post-doctoral in China Pharmaceutical University. His current research interests are biosensor and molecular diagnosis.
Yuanbiao Tu He is studying for Ph.D. degree in School of Engineering at China Pharmaceutical University. His current research interests are sensor and molecular diagnosis.
Haiping Wu She obtained her Ph.D. degree in microbiology and biochemical pharmacy from China Pharmaceutical University in 2010. Now she is a supervisor pharmacist in Huadong Research Institute for Medicine and Biotechnics. Her current research interests are biosensor and molecular diagnosis.
Hang Zhang He obtained his B.S. degree in bioengineering from China Pharmaceutical University in 2017. Now he is studying for M.S. degree in School of Engineering at China Pharmaceutical University. His current research interests are biosensor and molecular diagnosis.
HongHong Chen She obtained her B.S. degree in bioengineering from China Pharmaceutical University in 2018. Now she is studying for M.S. degree in School of Engineering at China Pharmaceutical University. Her current research interests are biosensor and molecular diagnosis.
Guohua Zhou He obtained his Ph.D. degree in analytical chemistry from Tsinghua University. Now he is a professor in Medical School of Nanjing University. His current research interests are molecular diagnosis and cancer screening.
Peng Wang He obtained his Ph.D. degree in biomedical engineering from Southeast University. Now he is a associate professor in School of Engineering at China Pharmaceutical University. His current research interests are sensor and molecular diagnosis.
Yueqing Gu She obtained her Ph.D. degree in physics from Nanjing University of Aeronautics and Astronautics. Now she is a professor in School of Engineering at China Pharmaceutical University major in biomedical engineering. Her current research interests are molecular imaging and nanomedicine.
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These authors contributed equally to this work.