High-sensitivity Analysis Of MicroRNA And Nucleic Acid Tool Enzymes Based On Terminal Extension-based Amplification Technology

The accurate detection of biomarkers such as microRNA and terminal deoxynucleotidyl transferase provides powerful tools for the rapid diagnosis of disease and prognosis evaluation.However,in complex matrix these biomarkers usually of ultralow concentrations.Currently,various amplification techniques have been developed for the high-sensitive detection of these biomarkers.Among them,the isothermal terminal extension-based amplification methods,including rolling circle amplification（RCA）and TdT mechanism,are emerging to be the most attractive and prominent methods for the analysis of various kinds of genetic biomarkers owing to the high reproducibility,flexible design,and easy operation.However,traditional terminal extension-based amplification technologies generally depend on linear amplifying mechanisms so that the amplification efficiency and detection sensitivity are greatly limited.To address the current limitations,this thesis has developed a series of innovative terminal extension-based amplification strategies,which have significantly improved the amplification efficiency and detection sensitivity,and are capable of detecting microRNA and TdT at single cell-level and even single-molecule level.（1）By rationally integrating the distinct advantages of RCA and loop-mediated isothermal amplification（LAMP）,an ultrasensitive RCA-LAMP method is devised for the detection of microRNA.In this design,the target microRNA can directly template the ligation of a padlock probe to trigger RCA-based terminal extension reaction,producing long and tandem amplification products.A rationally designed stem-loop primer（SLP）can bind with these tandem repeats one by one to initiate cascading extension and displacement reactions along the RCA products to generate a large amount of double stem-loop DNA structures,which are the essential starting materials to initiate subsequent efficient LAMP reaction.Different from the traditional RCA-based nucleic acid analysis assays,in this method each extension product of RCA can produce numerous double stem-loop DNA that can independently initiate exponential LAMP reaction.By conducting RCA and LAMP simultaneously accomplished in one step without additional sample transfer operations,even 10 aM of miRNA target can be unequivocally detectable,indicating the significantly improved amplification efficiency and detection sensitivity.（2）Inspired by the high-efficient RCA-LAMP assay,a novel method（TEA-LAMP）for the ultrasensitive detection of TdT activity has been proposed by combining the DNA terminal extension function of TdT and LAMP reaction.In this design,the elegant designed 3’-OH stem-loop primer（SLP-A）can only be extended in the presence of TdT to generate long poly（T）single-strand DNA.Another stem-loop primer（SLP-B）with an（A）-20 sticky tail will hybridize with poly（T）and initiate cascading extension and strand displacement reactions.By this means,each TdT extension product can produce numerous starting DNA structures of LAMP with a double stem-loop structure,which can independently initiate subsequent efficient LAMP reaction to realize the highly sensitive detection of TdT.The detection limit of TdT by using the TEA-LAMP strategy is as low as 2×10-8 U/μL,which is the to-date best performance for TdT detection to the best of our knowledge.Such an ultrahigh sensitivity enables the direct determination of TdT activity in individual single cells,which is of great significance in revealing the exact connection between cellular TdT heterogeneity and cell function as well as the major diseases evolution.In the meantime,by employing TdT as a co-factor,this strategy can also be applied to detecting other nucleic acid tool enzymes.（3）In the fourth chapter,a microRNA-induced hyperbranched RCA is designed by introducing a pair of primers.By combining T7 transcription and CRISPR-Casl2a-based amplification reaction,a triplex cascading signal amplification strategy（Cas-TCA）is developed for microRNA sensing.The addition of primer pair enables the full use of the substrate,increasing the amplification efficiency of RCA to accumulate a large number of double-stranded DNAs containing multiple T7 promoter sites for T7 RNA polymerase amplification and produce numerous pre-crRNA.The pre-crRNA is further processed by Cas 12a to produce mature crRNA,which can assemble with Cas 12a to recognize the dsDNA activators and activate the collateral cleavage activity of Cas 12a.As a result,the fluorophore/quencher-labeled ssDNA reporter will be cleaved with a high efficiency,resulting in a remarkable enhanced fluorescence signal.Different from the conventional CRISPR-Cas-based nucleic acid sensing systems that need pre-synthesis and purification steps to obtain crRNA,in this study,the pre-crRNA is generated by target-responsive amplification and further processed/recruited by Cas12a,without the involvement of any pre-synthesis and purification steps.This Cas-TCA method expands the CRISPR-Casbased sensing toolbox,showing great potential in CRISPR-Cas-based biological and biomedical studies.（4）The whole process of the Cas-TC A strategy includes multiple steps such as ligation,extension and transcription,which limits its application scenarios,especially when rapid detection is needed.To solve this issue,we has planned to combine all these steps to develop a simple and efficient one-step amplification strategy.However,experimental results have shown that these reaction systems are not compatible due to the complex composition of the buffer.Fortunately,we has found that under a constant temperature,the ligation reaction and T7 RNA transcription can be completed in one-step.On this basis,a strategy for the detection of microRNA based on one-step ligation and transcription has been established.In this strategy,the padlock probe,SplintR ligase,T7 RNA polymerse and buffer could participate the reaction simultaneously.In the presence of the target microRNA,the SplintR ligase initiates the padlock probe ligation reaction.Subsequently,the T7 RNA polymerase initiates transcription amplification（RCT）by using the ligated padlock probe as template.In addition,the transcription products contain the target sequence and can be used as a template for unligated padlock probe.This amplification mechanism can produce a large number of pre-crRNA.Finally,the sensitive detection of microRNA is realized based on the CRISPR-Cas12a detection system.