Bisulfite-free global DNA methylation analysis using multifunctional superparamagnetic nanomaterials

Abstract

DNA methylation naturally happens at the fifth carbon position of cytosine base within the CpG dinucleotides, plays a significant role in the numerous biological events, such as gene expression, cellular proliferation, embryonic developments, and chromosome instability. Aberrancies in DNA methylation pattern can lead to the genomic instability, resulting in the development of various human diseases including cancer, considered as one of the promising epigenetic (diagnostic and prognostic) biomarkers. Current research shows that abnormality in DNA methylation pattern presents a signature for disease diagnosis, therapeutic interventions, and prognosis of outcome. Therefore, current DNA methylation research has a major focus and needs for the development of easy, reliable and sensitive detection strategies. Throughout the last few decades, extensive research has been reported towards the quantification of DNA methylation in the mammalian genome. However, having their respective advantages in analytical performance and reliability, most of these strategies are confined to laboratory-based molecular biology techniques, such as real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarrays, and sequence-based methods. These methods largely require either bisulfite treatment (BT) or specific restriction enzyme digestion followed by a subsequence DNA amplification or sequencing. BT converts unmethylated cytosine to uracils leaving methylated cytosine unchanged, leveraging the methylated fragments distinguishable for detection. Low conversion, non-specific responses, false reading, longer assay time, amplification biased and complex chemistries significantly reducing the practice of BT and PCR amplification in current DNA methylation analysis. Hence, development of new analytical techniques without the BT steps and PCR amplification could be a valuable and out-of-bench tool for sensitive, low-cost and rapid platform to accomplish the emerging demand in genome-wide DNA methylation analysis in clinics. In this regard, bisulfite-free electrochemical biosensors have gained much attention in recent years as electrochemistry offers sensitive, cost-effective, portable, and simple biomolecules recognition readout. Parallel with electrochemistry, biosensors with optical readout enable direct real-time detection of biological molecules and are easily adaptable to multiplexing. Incorporation of electrochemical and optical readouts to bisulfite-free DNA methylation analysis is paving the way for translation of this important biomarker to standard patient care even resource-poor settings. This PhD thesis explores various electrochemistry along with colorimetric approach based sensitive, specific, rapid and inexpensive biosensor platform for bisulfitefree global DNA methylation analysis. Moreover, a series of commercial and in-house synthesised novel superparamagnetic nanomaterials were integrated to enhance the sensitivity and portability of the detection platform. A comprehensive literature review entailing detailed mechanism in DNA methylation pattern, association of DNA methylation with various human diseases, progress in DNA methylation biosensors techniques with a special emphasis on electrochemical and optical detection platforms have been reported. The challenges associated with current strategies have been outlined, and a great deal of recommendations also addressed to overcome the existing techniques. In the following chapters, four novel DNA methylation analysis strategies have reported based on colorimetry, electrochemistry and engineered nanostructures based inorganic enzymes (nanozymes) for the sensitive, rapid, and inexpensive analysis of global DNA methylation. First, based on the nucleic-acid affinity with the gold surface and methylation site-specific 5-mC antibody conjugated with horseradish peroxidase (HRP, natural enzyme), an amplification-free electrochemical and colorimetric assay was developed for the detection of global DNA methylation. In this strategy, the methylationsites to 5-mC antibody recognition were carried out on a screen-printed electrode surface and HRP catalysed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation were employed to read out the recognition event. The widely used colorimetry and ultra-sensitive chronoamperometry (i-t) were used to readout the DNA methylation pattern. Subsequent to the development of this proof-of-concept sensor, we replaced the commercial HRP enzyme with an in-house specially designed mesoporous iron-oxide for reading the methylation site recognition events in genomic DNA obtained from the oesophageal cancer cell lines. In this DNA biosensor, both the BT and PCR amplification were avoided to overcome the challenges associated with them and to achieve relatively simple and rapid DNA methylation detection. Prior to integration of mesoporous iron-oxides into the sensor platform, the peroxidase-like activity was explored and a colorimetric glucose sensor was developed. This intrinsic property was then combined with DNA methylation site-5-mC antibody immunocomplex and hence a sensitive colorimetric and electrochemical readout were reported for global DNA methylations. In the following chapter, a more sensitive assay was stated by utilising the coupling of enzymatic and electrocatalytic strengths of another natural (glucose oxidase) enzyme. In this assay, chronocoulometric (CC) charge measurement from redox mediator [Ru(NH3)6]3+ in the presence of glucose subtract via glucose oxidase enzyme was used for DNA methylation analysis. In our final readout strategies, we extended our approach towards an inexpensive and portable assay platform which enabled naked-eye, colorimetric and electrochemical interrogation of in-house synthesised porous graphene oxide-loaded iron oxide (GOFe2O3) via (TMB)/Nanozyme)-based colorimetric assay. All the readout platforms reported herein have shown excellent analytical performance with high sensitivity (limit of detection-LOD for global DNA methylation 5%) and specificity. The applicability of the assays was also demonstrated in different cancer cell line samples with high reproducibility. We envisage that this research efforts will contribute to the on-going development of simple, inexpensive and sensitive technology for global DNA methylation analysis towards commercial aspect of both the laboratory settings and resource-poor clinical settings.