Molecular Fishing Based Dissection of Epigenetic Markers in Live Cells

基於“分子釣魚”的活細胞表觀遺傳標誌物檢測技術

Student thesis: Doctoral Thesis

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Award date9 Dec 2021

Abstract

Cellular activities are under precise control of a variety of biomolecules. Understanding the fluctuation of biomolecules is of crucial importance for human health. As the DNA sequence could not provide the comprehensive information to completely understand the development of organisms and pathogenesis of diseases, the field of a new layer of regulation mechanism called epigenetics has undergone an enormous expansion in the last few years, especially in the areas related to RNA. Increasing evidence has demonstrated that non-coding RNA (e.g., microRNA) and RNA modification (e.g., RNA methylation) play crucial roles in biological processes. However, limited methods are applicable to profiling the dynamics of epigenetic regulation at RNA level. In this thesis, we tried to apply a molecular fishing-based approach to practical problems (e.g., subtype classification of disease), and then expand the target biomarkers to RNA methylation, making efforts to develop an effective and versatile platform to investigate epigenetic phenomena and thus deepen understandings of diseases.    The molecular fishing system is based on diamond nanoneedles that can capture molecules when functionalized with proper “baits”. We first applied the platform to liquid biopsy of microRNA, measuring the level of circulating microRNA in plasma directly. MicroRNAs embedded in lipid membrane or proteins were released into plasma, enabling direct enrichment of microRNAs on the surface of nanoneedles. When modified with size-selected p19 protein, the nanoneedles are capable of grasping double-stranded RNAs (dsRNAs) of specific length (20~22 bp) exclusively. The signals of isolated microRNAs then can be amplified by on-needle hybridization chain reaction and subsequently analyzed using confocal microscopy. Using this platform, we successfully detected microRNAs in plasma and then investigated the microRNA expression in patient samples.

To further explore the application of this platform, we next employed the platform to profile microRNA expression patterns in leukemia cell lines as a proof of concept of its feasibility to identify disease subtypes. With the help of centrifugal force, the nanoneedle can poke the cell membrane and capture miRNAs in cytoplasm. By combining the molecular fishing system and machine learning algorithms, we realized subtype stratification in acute leukemia cell mixtures and recognition of subpopulations in drug-treated cells.

To validate the feasibility and extendibility of the molecular fishing approach, we continued to seek a way to achieve RNA methylation analysis. As methylated mRNA has emerged importance in cellular activity and has drawn much attention from researchers, we focus on N6-methyladenosine (m6A) and N1-methyladesosine (m1A) in mRNA. By taking advantage of specific antibodies, RNA molecules with target chemical modifications can be pulled out from cytoplasm, ready for signal amplification with gene ID probes. Besides, a proximity ligation approach was developed to resolve dual methylation (m6A & m1A) on an individual mRNA segment. Using this method, we investigated the dynamics of mRNA methylation in mammalian cells under environmental stimuli.

In summary, our study demonstrates a highly efficient and flexible platform for microRNA and RNA methylation analysis based on molecular fishing. The employment of molecular fishing system to biomarkers detection has built a new bridge to unveil the mystery in epigenetics at RNA level, meanwhile providing a new way to investigate cell activity without damaging the cells.