Studies Of Fluorescent Biosensing New Methods For DNA Methyltransferase Activity Detection Based On DNA Probe

DNA methyltransferase(DNA MTase)is an epigenetic modification enzyme,which participates and plays an important role in many biological processes by catalyzing the DNA methylation process,such as regulating gene expression and development and so on.However,aberrant DNA MTase activity leads to the aberrant DNA methylation level which causes various types of diseases by the inactivation of tumor suppressor gene or silencing gene expression transcription,including developmental abnormalities,autoimmune diseases and many types of cancers.Thus,accurate and sensitive detection of DNA MTase activity has a guiding value in diagnosing the development stage and type of disease.At present,due to the high specificity of enzyme cleavage,the nuclease digestion-based method has attracted great attention in the detection of DNA MTase activity.In this method,the DNA probe as a recognition probe is methylated by catalyzing of DNA MTase,and then is cleaved by nuclease digestion to achieve the transduction of the target DNA MTase.Subsequently,a variety of techniques for signal output are assisted to achieve the detection of DNA MTase activity.Among them,fluorescent technique-assisted biosensing methods have been used for the detection of DNA MTase activity with simple,rapid and sensitive properties.However,there are still some problems to be solved,that is,the low amplification efficiency,partially enclosed manner of recognition probe and low transduction efficiency have limitations to improve the existing sensitivity.And the need of excess auxiliary probe during the detection process causes signal interference and increases the complexity of their own design.DNA MTase has been used as the research model.In view of the above problems in the current detection methods,this paper has carried out the related research.This paper is dvided into six chapters,and the main content of each chapter is summarized as follows:Chapter one is the introduction section,which mainly summarizes the definition,function,category and research significance of DNA MTase.And the existing methods for the detection of DNA MTase activity and the problems in them are summarized.In addition,the principle,category and biological application of fluorescent biosensing methods are also briefly summarized.In chapter two,based on strand displacement amplification and DNAzyme amplification reactions,we have developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity.In the double-stranded recognition probe,the primer and template of strand displacement amplification and synthesizing template of DNAzyme are introduced to effectively combine strand displacement amplification and DNAzyme amplification reactions.After the enzyme catalysis,the recognition probe maintains the original double-stranded structure unchanged.The short strand acts as the primer of strand displacement amplification,and the long strand acts as the template,which initiate strand displacement amplification reaction with the aid of enzyme.Subsequently,the free DNAzyme sequences are synthesized by the above amplification reaction,due to the synthesizing template of DNAzyme in recognition probe.Then,the free DNAzyme sequences cyclically cleave the molecular beacon substrates,releasing DNA fragments with fluorophores.The fluorescent signal is recovered to achieve the detection of DNA MTase activity.This strategy combines two amplification reactions,greatly improving the amplification efficiency and ensuring a high sensitivity of the detection strategy with the corresponding detection limit of 0.0082 U/mL.In chapter three,based on strand displacement amplification and exponential rolling circle amplification reactions,we have designed a loop-mediated cascade amplification strategy for ultrasensitive and accurate detection of DNA MTase activity.In this design,the long stem-loop probe has been used as recognition probe.After the methylation recognition event,the loop and adjacent stem is released as trigger strand for subsequent processes.The long stem of the recognition probe has two effects:on the one hand,maintaining the stability of the probe or ensuring the effective formation of the probe,on the other hand,effectively enclosing the trigger strand to prevent the nonspecific amplification caused by probe leakage.The released trigger strand hybridizes with hairpin probe,and then triggers the strand displacement amplification reaction,generating the primers.Subsequently,the primers hybridize to padlock probes with three cleavage sites,and then trigger exponential rolling circle amplification reaction,obtaining a significantly enhanced fluorescent signal.Combined with the high amplification efficiency of cascade amplification reaction and the high stability of long stem-loop probe,the detection limit of DNA MTase activity is reduced to 8.1 ×10-5 U/imL?In chapter four,we have developed a multiple-closed primers-mediated rolling circle amplification strategy to detect DNA MTase activity by the effective closed effect of the double-loop probe on primers.We have designed a double-loop probe with multiple primers as the recognition probe.Under the action of DNA MTase,the multiple transduction strands are released and act as primers(entire primers or split primers)for the subsequent rolling circle amplification reaction.The high transduction efficiency of 1:N transduction mode can effectively improve detection sensitivity of DNA MTase activity.And by changing the number of primers in double-loop probe,the controllability of the enzyme acticity detection is achieved.In addition,with the increasing of closed primers,the detection sensitivity is accordingly improved.And for each additional primer,the detection limit is about reduced by an order of magnitude.The high transduction efficiency of the 1:N transduction mode and the high amplification efficiency of rolling circle amplification reaction are combined to effectively improve the detection sensitivity of DNA MTase activity,with the detection limit as low as 0.0085 U/mL.Compared with entire primers,the background signal is lower and detection limit is slightly reduced due to the improved specificity of split primers.In chapter five,based on the conformation transition mechanism,we have designed an integrated conformational variable probe-mediated strand displacement amplification strategy for DNA MTase activity detection.We have designed an integrated conformational variable probe as the recognition probe,which contains the primer and template of strand displacement amplification reaction.When DNA MTase is absent,the primer and template are closed in recognition probe.However,when DNA MTase is present,it catalyzes the methylation event of the probe which is then cleaved by nuclease,producing a new hairpin probe with long loop and short stem.The hairpin probe is unstable and undergoes a conformation transition to activate the primer and template for triggering strand displacement amplification reaction.In this design,the recognition probe combines recognition,transduction,and signal output elements to greatly simplify the difficulty of the probe design.The simplicity of probe design and the strong amplification ability of the amplification process can be beneficial to the simple and sensitive detection of DNA MTase activity with the detection limit of 0.063 U/mL.Chapter six is the conclusion section,which mainly contains the work summary and foreground prospect of this paper.