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An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling
Published on: December 18, 2017
Label-Free Raman Observation of TET1 Protein-Mediated Epigenetic Alterations in DNA
Xiaojun Luo1, Lijuan Jiang1, Tuli Kang1
1Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, College of Chemistry and Materials Science , Nanjing Normal University , Nanjing , Jiangsu 210097 , P.R. China.
Abstract:
Epigenetic modifications of DNA are known to modulate gene activity and expression and are believed to result in genetic diseases, such as cancer. Four modified cytosines were discovered in mammalian genomes: 5-methycytoine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). They are regarded as DNA epigenetic markers and play key roles in the regulation of the dynamic balance between DNA methylation and demethylation. Although detection approaches toward 5mC are ubiquitous, few assays have reported the simultaneous determination of all four modified cytosines as well as monitoring of their dynamic alterations. Here, we developed a label-free surface enhanced Raman spectroscopy (SERS)-based method for directly sensing the four DNA modifications by using a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface as the substrate. This method is based on identifying SERS spectral features resulting from DNA base modifications. Our study shows that 5mC, 5hmC, 5fC, and 5caC exhibit distinct Raman spectroscopic signatures at 785, 660, 1450, and 1680 cm-1, respectively. Moreover, the developed method can be used for tracking of the dynamic alterations among these four modified cytosines in DNA mediated by the ten-eleven translocation (TET) protein. The dynamic stepwise conversion from 5mC into 5hmC, 5fC, and 5caC is further demonstrated to be a typical three-step consecutive reaction with rate constants of 0.6, 0.25, and 0.15 min-1, respectively, which has not been achieved before via a SERS-based method.
Insights
This study introduces a novel label-free SERS method to detect four DNA epigenetic markers: 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). The method successfully tracks dynamic alterations between these modified cytosines in DNA.
Area of Science:
- Epigenetics and Molecular Biology
- Biophysical Chemistry
- Genomics
Background:
- DNA epigenetic modifications, including four key cytosine variants (5mC, 5hmC, 5fC, 5caC), regulate gene expression and are implicated in diseases like cancer.
- While 5mC detection is common, simultaneous analysis of all four modified cytosines and their dynamic changes remains challenging.
- These modified cytosines are crucial epigenetic markers involved in maintaining the balance of DNA methylation and demethylation.
Purpose of the Study:
- To develop a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for direct sensing of four distinct DNA modified cytosines.
- To enable simultaneous detection and monitoring of dynamic alterations among 5mC, 5hmC, 5fC, and 5caC.
- To investigate the kinetics of DNA modification conversions mediated by TET proteins.
Main Methods:
- Utilized a plasmonic gold nanohole array (PGNA) substrate for Surface-Enhanced Raman Spectroscopy (SERS).
- Employed a label-free approach to identify distinct SERS spectral signatures of 5mC, 5hmC, 5fC, and 5caC.
- Monitored the dynamic conversion of these modified cytosines in DNA, particularly in the presence of ten-eleven translocation (TET) proteins.
Main Results:
- Successfully identified unique SERS spectral signatures for 5mC (785 cm⁻¹), 5hmC (660 cm⁻¹), 5fC (1450 cm⁻¹), and 5caC (1680 cm⁻¹).
- Demonstrated the capability of the SERS method to track dynamic alterations among the four modified cytosines.
- Quantified the stepwise conversion of 5mC to 5hmC, 5fC, and 5caC as a three-step consecutive reaction with determined rate constants.
Conclusions:
- The developed label-free SERS method provides a sensitive and direct approach for simultaneous detection of four key DNA epigenetic markers.
- This technique offers a powerful tool for studying the dynamic regulation of DNA methylation and demethylation.
- The kinetic analysis of TET-mediated conversions provides novel insights into the mechanisms of active DNA demethylation.
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