Copper-Free Click Chemistry-Mediated Assembly of Single Quantum Dot Nanosensor for Accurately Monitoring Locus-Specific m6A in Cancer Cells†
Yue-Ying Li
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
These authors contributed equally.
Search for more papers by this authorNing-Ning Zhao
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
These authors contributed equally.
Search for more papers by this authorYi-Xuan Geng
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
These authors contributed equally.
Search for more papers by this authorQian Han
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, 211189 China
Search for more papers by this authorJian-Ge Qiu
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
Search for more papers by this authorCorresponding Author
Bing-Hua Jiang
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorCorresponding Author
Zi-Yue Wang
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorCorresponding Author
Chun-Yang Zhang
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, 211189 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorYue-Ying Li
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
These authors contributed equally.
Search for more papers by this authorNing-Ning Zhao
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
These authors contributed equally.
Search for more papers by this authorYi-Xuan Geng
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
These authors contributed equally.
Search for more papers by this authorQian Han
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, 211189 China
Search for more papers by this authorJian-Ge Qiu
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
Search for more papers by this authorCorresponding Author
Bing-Hua Jiang
Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University, Zhengzhou, Henan, 450052 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorCorresponding Author
Zi-Yue Wang
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong, 250014 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorCorresponding Author
Chun-Yang Zhang
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, 211189 China
E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this authorDedicated to the Special Issue of Single-Molecule Measurement and Imaging.
Comprehensive Summary
N6-methyladenosine (m6A) plays an important role in embryogenesis, nuclear export, transcription splicing, and protein translation control. Herein, we demonstrate a copper-free click chemistry-mediated assembly of single quantum dot (QD) nanosensor for accurately monitoring locus-specific m6A in cancer cells. The m6A-sensitive endoribonuclease MazF can digest the unmethylated A-RNA, and the intact m6A-RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid, initiating the click chemistry to generate probe a–b ligation product via first tandem ligation detection reaction (LDR) cycle. Subsequently, DNA probes c and d can hybridize with the probe a–b ligation product to generate the probe c–d ligation product via second LDR cycle. Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin-/fluorophore-modified probe c–d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer (FRET) between QD and Cy5. This assay can be homogenously performed without the involvement of copper catalyst, m6A-specific antibody, radioactive labeling, ligase enzyme, enzymatic reverse transcription, and next-generation sequencing. Moreover, it can discriminate even 0.01% m6A level in complex samples and accurately measure cellular m6A-RNA expression, providing a promising avenue for clinical diagnostics and biomedical research.
Supporting Information
| Filename | Description |
|---|---|
| cjoc202300595-sup-0001-supinfo.pdfPDF document, 667.8 KB |
Appendix S1: Supporting Information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1 Imam, H.; Khan, M.; Gokhale, N. S.; McIntyre, A. B. R.; Kim, G. W.; Jang, J. Y.; Kim, S. J.; Mason, C. E.; Horner, S. M.; Siddiqui, A. N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 8829–8834.
- 2 Lai, G. Q.; Zhou, L. L.; Yang, C. G. RNA Methylation mA: A New Code and Drug Target? Chin. J. Chem. 2020, 38, 420–421.
- 3 Hong, T.; Yuan, Y.; Chen, Z.; Xi, K.; Wang, T.; Xie, Y.; He, Z.; Su, H.; Zhou, Y.; Tan, Z. J.; Weng, X.; Zhou, X. Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution. J. Am. Chem. Soc. 2018, 140, 5886–5889.
- 4 Li, Y.; Sun, Y.; Yang, W.; Yang, L.; Su, M.; Fang, L.; Zheng, J.; Yuan, R.; Liang, W. A novel photoelectrochemical strategy for sequence-spot bispecific analysis of N6-methyladenosine modification based on proximity ligation-triggered cascade amplification. Anal. Chim. Acta 2023, 1265, 341287.
- 5 Pu, Q. L.; Ye, Y. Y.; Hu, J.; Xie, C.; Zhou, X.; Yu, H. Y.; Liao, F. L.; Jiang, S.; Jiang, L. S.; Xie, G. M.; Chen, W. X. XNA probe and CRISPR/ Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification. Talanta 2023, 252, 123754.
- 6 Yin, H. S.; Zhou, Y. L.; Yang, Z. Q.; Guo, Y. L.; Wang, X. X.; Ai, S. Y.; Zhang, X. S. Electrochemical immunosensor for N6-methyladenosine RNA modification detection. Sens. Actuator B-Chem. 2015, 221, 1–6.
- 7 Price, A. M.; Hayer, K. E.; McIntyre, A. B. R.; Gokhale, N. S.; Abebe, J. S.; Della Fera, A. N.; Mason, C. E.; Horner, S. M.; Wilson, A. C.; Depledge, D. P.; Weitzman, M. D. Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing. Nat. Commun. 2020, 11, 6016.
- 8 Zhao, N. N.; Liu, Y. Z.; Zhang, L. F.; Liu, W. J.; Zou, X. R.; Xu, Q. F.; Zhang, C. Y. Construction of Multiple DNAzymes Driven by Single Base Elongation and Ligation for Single-Molecule Monitoring of FTO in Cancer Tissues. Anal. Chem. 2023, 95, 12974–12981.
- 9 Zhao, N. N.; Zhang, X. Y.; Zou, X. R.; Zhang, Y.; Zhang, C. Y. Controllable assembly of dendritic DNA nanostructures for ultrasensitive detection of METTL3-METTL14 m6A methyltransferase activity in cancer cells and human breast tissues. Biosens. Bioelectron. 2023, 228, 115217.
- 10 Liu, L.; Kong, Y. Q.; He, L.; Wang, X. X.; Wang, M. M.; Xu, H. J.; Yang, C. G.; Su, Z.; Zhao, J.; Mao, Z. W.; Huang, Y.; Liu, H. K. A Rhein-Based Rh(III) Arene Complex with Anti-tumor Cell Proliferative Activity Inhibits RNA Demethylase FTO. Chin. J. Chem. 2022, 40, 1156–1164.
- 11 Harcourt, E. M.; Ehrenschwender, T.; Batista, P. J.; Chang, H. Y.; Kool, E. T. Identification of a selective polymerase enables detection of N(6)-methyladenosine in RNA. J. Am. Chem. Soc. 2013, 135, 19079–19082.
- 12Li, W.-x.; Jiang, S.; Liu, W.-j.; Zhang, C.-y. RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues. Anal. Chim. Acta 2023, 1260, 341208.
- 13 Sommer, S.; Salditt-Georgieff, M.; Bachenheimer, S.; Darnell, J. E.; Furuichi, Y.; Morgan, M.; Shatkin, A. J. The methylation of adenovirus-specific nuclear and cytoplasmic RNA. Nucleic Acids Res. 1976, 3, 749–765.
- 14 Narayan, P.; Ludwiczak, R. L.; Goodwin, E. C.; Rottman, F. M. Context effects on N6-adenosine methylation sites in prolactin mRNA. Nucleic Acids Res. 1994, 22, 419–426.
- 15 Liebich, H. M.; Xu, G.; Di Stefano, C.; Lehmann, R.; Häring, H. U.; Lu, P.; Zhang, Y. Analysis of normal and modified nucleosides in urine by capillary electrophoresis. Chromatographia 1997, 45, 396–401.
- 16 Bodi, Z.; Fray, R. G. Detection and Quantification of N6-Methyladenosine in Messenger RNA by TLC. Methods Mol. Biol. 2017, 1562, 79–87.
- 17 Yin, H. S.; Wang, H. Y.; Jiang, W. J.; Zhou, Y. L.; Ai, S. Y. Electrochemical immunosensor for N6-methyladenosine detection in human cell lines based on biotin-streptavidin system and silver-SiO2 signal amplification. Biosens. Bioelectron. 2017, 90, 494–500.
- 18 Selmi, T.; Lanzuolo, C. Driving Chromatin Organisation through N6-methyladenosine Modification of RNA: What Do We Know and What Lies Ahead? Genes 2022, 13, 340.
- 19 Dominissini, D.; Moshitch-Moshkovitz, S.; Schwartz, S.; Salmon-Divon, M.; Ungar, L.; Osenberg, S.; Cesarkas, K.; Jacob-Hirsch, J.; Amariglio, N.; Kupiec, M.; Sorek, R.; Rechavi, G. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 2012, 485, 201–206.
- 20 Wan, Y. Z.; Tang, K.; Zhang, D. Y.; Xie, S. J.; Zhu, X. H.; Wang, Z. G.; Lang, Z. B. Transcriptome-wide high-throughput deep m6A-seq reveals unique differential m6A methylation patterns between three organs in Arabidopsis thaliana. Genome Biol. 2015, 16, 272.
- 21 Hashimoto, M.; Hupert, M. L.; Murphy, M. C.; Soper, S. A.; Cheng, Y. W.; Barany, F. Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low- abundant DNA point mutations. Anal. Chem. 2005, 77, 3243–3255.
- 22 Wang, L. Y.; He, R. Y.; Lv, B.; Yu, X.; Liu, Y.; Yang, J.; Li, W. Q.; Wang, Y.; Zhang, H.; Yan, G. B.; Mao, W. X.; Liu, L. L.; Wang, F.; Ma, L. X. Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV. Talanta 2021, 227, 122154.
- 23 Ma, F.; Liu, H.; Li, C. C.; Zhang, C. Y. A simple and isothermal ligase-based amplification approach based on a ligation-activated cleavage reaction. Chem. Commun. 2018, 54, 12638–12641.
- 24 Wang, Z. C.; Li, L.; Kuang, Y. Q.; Yao, J.; Xu, F. F.; Chen, Y. Simultaneous quantification of multiple single nucleotide variants in PIK3CA ctDNA using mass-tagged LCR probe sets. Talanta 2023, 258, 124426.
- 25 Xiao, Y.; Wang, Y.; Tang, Q.; Wei, L. H.; Zhang, X.; Jia, G. F. An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N6-Methyladenosine Modification. Angew. Chem. Int. Ed. 2018, 57, 15995–16000.
- 26 Chapman, R.; Stenzel, M. H. All Wrapped up: Stabilization of Enzymes within Single Enzyme Nanoparticles. J. Am. Chem. Soc. 2019, 141, 2754–2769.
- 27 Ma, F.; Li, C. C.; Zhang, C. Y. Nucleic acid amplification-integrated single-molecule fluorescence imaging for in vitro and in vivo biosensing. Chem. Commun. 2021, 57, 13415–13428.
- 28 Zheng, J. N.; Mao, Y.; Feng, S.; Tian, R. J. Combining Metabolic Alkyne Labeling and Click Chemistry for Secretome Analysis of Serum-Containing Conditioned Medium. Chin. J. Chem. 2021, 39, 1843–1848.
- 29 Xiang, W. W.; Zhang, Z. J.; Weng, W. Q.; Wu, B. D.; Cheng, J.; Shi, L.; Sun, H. W.; Gao, L.; Shi, K. Q. Highly sensitive detection of carcinoembryonic antigen using copper-free click chemistry on the surface of azide cofunctionalized graphene oxide. Anal. Chim. Acta 2020, 1127, 156–162.
- 30 Imanishi, M.; Tsuji, S.; Suda, A.; Futaki, S. Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease. Chem. Commun. 2017, 53, 12930–12933.
- 31 Yim, S. H.; Cha, H. J.; Park, S. J.; Yim, Y.; Woo, J. S.; Min, D. H. A fluorescent nanobiosensor for the facile analysis of m6A RNA demethylase activity. Chem. Commun. 2020, 56, 4716–4719.
- 32 Wang, Z. Y.; Li, P.; Hu, J. P.; Xu, Q. F.; Zhang, C. Y. Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N6-Methyladenosine in Cancer Cells and Tissues. Anal. Chem. 2023, 95, 5454–5462.
- 33 Xin, H. B.; Namgung, B.; Lee, L. P. Nanoplasmonic optical antennas for life sciences and medicine. Nat. Rev. Mater. 2018, 3, 228–243.
- 34 Zhang, Y. J.; Yan, S.; Chen, Z. X.; Jiang, X.; Rao, S.; Jiang, Z. R.; Qin, S. S.; Zhou, X.; Du, Y. H. Visually Intracellular Detection of Telomerase Activity Based on DNA Strand Displacement Reaction and Gold Nanoparticle Labeling. Chin. J. Chem. 2022, 40, 693–698.
- 35 Li, X. L.; Wang, Y.; Song, A.; Zhang, M. H.; Chen, M. N.; Jiang, M.; Yu, S. S.; Wang, R. Z.; Xing, L. B. An Artificial Light-Harvesting System with Tunable Fluorescence Color in Aqueous Sodium Dodecyl Sulfonate Micellar Systems for Photochemical Catalysis. Chin. J. Chem. 2021, 39, 2725–2730.
- 36 Su, D. D.; Li, H. X.; Yan, X.; Lin, Y. H.; Lu, G. Y. Biosensors based on fluorescence carbon nanomaterials for detection of pesticides. Trac-Trends Anal. Chem. 2021, 134, 116126.
- 37 Zhou, J.; Yang, Y.; Zhang, C. Y. Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 2015, 115, 11669–11717.
- 38 Hildebrandt, N.; Spillmann, C. M.; Algar, W. R.; Pons, T.; Stewart, M. H.; Oh, E.; Susumu, K.; Diaz, S. A.; Delehanty, J. B.; Medintz, I. L. Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications. Chem. Rev. 2017, 117, 536–711.
- 39 Zhang, Y.; Hu, J. P.; Zou, X. R.; Ma, F.; Qiu, J. G.; Zhang, C. Y. Integration of single-molecule detection with endonuclease IV-assisted signal amplification for sensitive DNA methylation assay. Chem. Commun. 2021, 57, 2073–2076.
- 40 Hu, J.; Pan, L. Y.; Li, Y. Y.; Zou, X. R.; Liu, B. J.; Jiang, B. H.; Zhang, C. Y. Deacetylation-activated construction of single quantum dot-based nanosensor for sirtuin 1 assay. Talanta 2021, 224, 121918.
- 41 Zhang, C. L.; Ding, C. P.; Xiang, D. S.; Li, L.; Ji, X. H.; He, Z. K.; Xian, Y. Z. DNA Functionalized Fluorescent Quantum Dots for Bioanalytical Applications. Chin. J. Chem. 2016, 34, 317–325.
- 42 Zhong, Z.-D.; Xie, Y.-Y.; Chen, H.-X.; Lan, Y.-L.; Liu, X.-H.; Ji, J.-Y.; Wu, F.; Jin, L.; Chen, J.; Mak, D. W.; Zhang, Z.; Luo, G.-Z. Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing. Nat. Commun. 2023, 14, 1906.
- 43 Lee, J. Y.; Kim, M.; Lee, C.; Kim, D. N. Characterizing and Harnessing the Mechanical Properties of Short Single-Stranded DNA in Structured Assemblies. ACS Nano 2021, 15, 20430–20441.
- 44 Jouchet, P.; Cabriel, C.; Bourg, N.; Bardou, M.; Pous, C.; Fort, E.; Leveque-Fort, S. Nanometric axial localization of single fluorescent molecules with modulated excitation. Nat. Photonics 2021, 15, 297–304.
- 45 Hu, J.; Li, Y. Y.; Li, Y.; Tang, B.; Zhang, C. Y. Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GIcNAc Transferase Activity. Anal. Chem. 2017, 89, 12992–12999.
- 46 Ou, X.; Pu, Q.; Sheng, S.; Dai, T.; Gou, D.; Yu, W.; Yang, T.; Dai, L.; Yang, Y.; Xie, G. Electrochemical competitive immunodetection of messenger RNA modified with N6-methyladenosine by using DNA- modified mesoporous PtCo nanospheres. Mikrochim. Acta 2019, 187, 31.
