Construction Of Biosensor Technology And Application In DNA And RNA Methylation Analysis

Research background:Gene methylation is a major modification of the epigenetic,which refers to the adjunction of a methyl group（-CH₃）to specific bases under the action of corresponding methyltransferase without changing the oligonucleotide sequence of the gene,mainly including DNA methylation,RNA methylation,etc.In mammals,DNA methylation mainly occurs in the formation of 5-methyl Cytosine（m⁵C）,while RNA methylation mainly includes m⁶A,m⁷G,m⁵C,m¹A,Um and other types,among which m⁶A and m⁵C are the most abundant modification types in most eukaryotes.Studies have shown that methylation plays a crucial role in the growth and development of mammals by inhibiting or promoting the expression of genes and thus causing changes in biological functions.At the same time,the bias of m⁶A and m⁵C modification levels also have an remarkable influence on the control of the biological clock,the maintenance of pluripotency of embryonic stem cells,and the development of many complex human diseases,such as leukemia,breast cancer,lung cancer,cardiovascular disease,autoimmune disease,metabolic disease,neuronal disease,etc.The abnormal methylation level usually appears earlier than the malignant proliferation of cells.Therefore,the abnormal methylation level of genomes has become a biomarker for epigenetic disease diagnosis and treatment.Such as RASSF1A,PRDM1,HOXA9 gene methylation has become markers for early diagnosis,thus to build appropriate clinical detection technology to analyze DNA and RNA methylation level is of great significance.At present,the techniques for DNA methylation analysis mainly include bisulfite pretreatment,high performance liquid chromatography（HPLC）or mass spectrometry,restriction enzyme digestion pretreatment（MSRE）and so on.RNA methylation detection methods mainly include methylated RNA immunoprecipitation sequencing（MERIP-seq）,site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography（SCARLET）,single nucleotide resolution cross-linking and immunoprecipitation（miCLIP）,etc.Although each of them has its own advantages,they are difficult to be popularized and used in clinical laboratories because of the complicated analysis process,huge data processing after testing and high technical requirements.Therefore,the establishment of a detection technique with good specificity,simple operation and easy popularization has become an urgent need for the early diagnosis of clinical epigenetic disorders and the plan selection of tumor demethylation therapy.Research objectives:In view of the existing problems of DNA methylation analysis technology,this study aims to construct DNA single-mode methylation analysis technology with clinical application prospect by using the advantages of electrochemical biosensor technology,such as convenient operation,fast analysis speed and easy clinical laboratory popularization,as well as the characteristics of high catalytic efficiency,simple preparation,strong selectivity,good stability and good biocompatibility of DNA nano-self-assembly catalysis technology.Based on the DNA single-mode methylation analysis technology,in order to further improve the sensitivity and specificity of methylation detection in the early diagnosis of disease.A RNA multimodal methylation analysis technology was constructed to synchronization analyze m⁶A and m⁵C modification sites in RNA.Research methods:1.Based on 3D matrixed DNA self-nanocatalyzer to analyze single-mode DNA methylation sites（1）Preparation and validation of 3D matrixed DNA self-nanocatalyzer.（2）The preparation of nanocomposites by using AuNPs and GO.（3）Based on 3D matrixed DNA self-nanocatalyzer to construct an electrochemical sensing platform analyze the target with different number of methylation sites.（4）Optimize the conditions and analyze the performance of the electrochemical sensing platform.2.Based on 3D matrixed DNA self-nanocatalyzer and AuNPs/g-C₃N₄@rGO to analyze single-mode DNA methylation abundance（1）Preparation and characterization of AuNPs/g-C₃N₄@rGO nanocomposites.（2）The construction of electrochemical sensing platform and the comparison of different signal amplification strategies.（3）Performance analysis of electrochemical sensing platform.（4）Analysis of clinical simulated specimens.3.Photoelectrochemical biosensor technology for multimodal RNA methylation analysis based on immune competition and DNA origami structure（1）Construction of immune competition system.（2）Construction and verification of H-type nano-scaffolds and DNA Walker system.（3）Construction and characterization of simple DNA origami.（4）Assembly of photoelectrochemical biosensor platform and optimization of experimental conditions.（5）Performance analysis of photoelectrochemical biosensor platform.Research results:1.Successfully constructed DNA single-mode methylation quantitative analysis technology based on the 3D matrixed DNA self-nanocatalyzer,the method can be detected the largest number of DNA methylation site was 7 under the optimal experimental conditions,the relationship between electrochemical signals and the number for methylation site was I（μA）=1.4272+1.7830*N,R²=0.9928,which proved that it can realize more quantitative analysis of methylation site.2.AuNPs/g-C₃N₄@rGO nanocomposites can be further improved the performance of the analysis technology.The strategy based on 3D matrixed DNA self-nanocatalyzer and AuNPs/g-C₃N₄@rGO quantitative analysis of DNA methylation has high sensitivity with the detection limit of 8.6 a M.The linear equation is I（μA）=8.3047+0.3431*log C（R²=0.994）,with a linear range from 10^-17-10^-8 M,which is superior to other strategies proposed in other studies,and has a prospect of clinical application.3.A photoelectrochemical biosensor technology for quantitative analysis of RNA multimodal methylation based on DNA origami technology has been successfully constructed.This technology has good specificity and sensitivity,and can simultaneously analyze m⁶A-RNA and m⁵C-RNA with the detection limits of 1.96 f M and 7.37 p M,respectively,which is superior to other reported analysis technology and has great application prospect.Conclusion:In this study,an electrochemical biosensor technology for quantitative analysis of DNA single-mode methylation was successfully constructed,which have good stability,repeatability,specificity and clinical application prospect.Based on this technology,the photoelectrochemical biosensor technology can simultaneously analyze m⁶A-RNA and m⁵C-RNA was proposed and successfully constructed for the first time,which provides a new method for the analysis of RNA methylation,and plays an important role in the early diagnosis,treatment and prognosis evaluation of methylation disorders.