Label-Free Raman Observation of TET1 Protein-Mediated Epigenetic Alterations in DNA

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), 5hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5carboxycytosine (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.

A Review of Surface-Enhanced Raman Spectroscopy on Potential Clinical Applications Towards Diagnosing Colorectal Cancer

“…The focus of this study was to highlight the high sensitivity of SERS for single-molecule detection in minute changes of a SERS peak due to particular DNA methylation. Other instances of using SERS for DNA methylation can also be found in these studies [50][51][52]. The specifics of the SERS detection of DNA epigenetic is focusing on the vibrational assignment of the cytosine and guanine nucleotides, where ring breathing, CH/ CH2 deformation, and C=O stretching modes are picked up with different intensities.…”

“…The specifics of the SERS detection of DNA epigenetic is focusing on the vibrational assignment of the cytosine and guanine nucleotides, where ring breathing, CH/ CH2 deformation, and C=O stretching modes are picked up with different intensities. In addition, the redshifting of vibrational modes can also indicate specific modifications [52].…”

Metallic Plasmonic Array Structures: Principles, Fabrications, Properties, and Applications

“…[182][183][184] These problem can be partially overcome by introducing an optical cavity into the structure to form a gap-free SERS substrate. [74,111,[185][186][187] As shown in Figure 6d-i, Yu et al fabricated a Au nanohole-based gap-free SERS substrate by using EBL method. [185] The enhanced near-field of the structure originates from the coupling of F-P cavity and LSPR mode of nanohole and is mainly localized at the edge of the top nanoholes (Figure 6d-ii).…”

“…[74,111,[185][186][187] As shown in Figure 6d-i, Yu et al fabricated a Au nanohole-based gap-free SERS substrate by using EBL method. [185] The enhanced near-field of the structure originates from the coupling of F-P cavity and LSPR mode of nanohole and is mainly localized at the edge of the top nanoholes (Figure 6d-ii). Since the near-field enhancement is not from the narrow gap, the SERS enhancement of the array substrate is insensitive to the slight variation of the gap size.…”

“…Since the near-field enhancement is not from the narrow gap, the SERS enhancement of the array substrate is insensitive to the slight variation of the gap size. This feature greatly facilitates the preparation of the plasmonic array, promoting their applications in DNA detection (Figure 6d-iii), [185,188] pH sensing, [189] pharmaceutical analysis, [190] etc. This kind of structures can also be prepared on a flexible substrate.…”