Fluorescence Analysis Of MicroRNAs Based On Transcriptional Amplification Mechanisms

MicroRNAs(MiRNAs)are a type of single-stranded small non-coding RNAs with 18?25 nucleotides in length,which play critical roles in regulating gene expression in both animals and plants.Some recent evidence has manifested that miRNAs derived from food plants can be found in mammalian and these miRNAs might have the ability to regulate the expression of animal and even human target genes.What is more,the abnormal expression of miRNAs have been found to be closely related to many human diseases,such as cancer.So miRNAs are deemed to be important biomarkers for clinical cancer diagnosis,prognosis,and therapy.Therefore,accurate and highly sensitive detection of miRNAs is of great importance,and how to develop sensitive analytical methods that can detect both animal miRNAs and plant miRNAs through simple design is still remain challenging.In this thesis,two analytical methods coupled with miRNAtemplated ligation and T7 RNA polymerase-based transcription amplification were developed to achieve high-sensitive detection of miRNAs.The main contents are as follows:In the second chapter of the dissertation,a plant miRNA detection method was proposed coupled with cycling click chemistry ligation and poly(T)-promoted transcription amplification.In this design,the target miRNA regulated the thermalinduced cycling click chemistry ligation of two DNA probes,which acted as the first-step amplification.The ligated DNA strands containing T7 promoter sequences bound to the surface of magnetic beads(MBs)via a streptavidin-biotin interaction and then hybridize with the poly(T)transcription templates.Initiated by the T7 RNA polymerase,the in vitro transcription was templated by poly(T)downstream of the T7 promoter sequence and produced numerous RNAs for the second-step amplification.Surprisingly,for the first time we find that the poly(T)template assisted in producing ultra-long RNA strands and exhibited much-improved transcription efficiency compared to the other reported templates.This poly(T)sequence-dependent transcription behavior of the T7 RNA polymerase has vastly improved the transcription efficiency as well as the detection performance.After the MBs have been removed by magnetic separation,the pure RNAs can be easily detected after adding the RNA intercalating dye RiboGreen.The two-step signal amplification strategy,which integrates the efficient cycling click chemistry ligation and poly(T)-promoted transcription amplification,provides a new way for the detection of both plant miRNAs and animal miRNAs.In the third chapter of this paper,a new miRNA biosensing mechanism was developed based on Cas12a self-powered crRNA(CRISPR RNA)recruiting(Cas12a-SCR).In this Cas12a-SCR system,miRNA-mediated enzymatic ligation of padlock probes can specifically trigger rolling circle transcription(RCT)to produce a long single-strand RNA(ssRNA)with numerous pre-crRNA(precursor crRNA)repeats,which can be trimmed and recruited actively by Casl2a.Then the trimmed mature crRNA and Cas12a bound with each other to form Cas12a-crRNA complexes.After that,Cas12a-crRNA will be activated by double-strand DNA(dsDNA)activators to efficiently nonspecific cleave single-strand DNA(ssDNA)reporter and release strong fluorescence signal.The utilization of Exonuclease I(Exo I)remarkably suppressed the nonspecific background,thus endowing the Cas12a-SCR a high sensitivity for miRNA analysis with a low detection limit of 0.47 amol.What is more,the miRNA-initiated RCT amplification products acted as the crRNA fuel but not the targeting sequences of Casl2a-crRNA,which also relieved the stringent requirement of PAM(protospacer-adjacent motif)site in the dsDNAtarget sequence and thus greatly expanded the universality of the Cas12a-SCR towards different nucleic acid sequences.The two novel methods of miRNA fluorescence sensing established in this paper have the universality for the detection of animal miRNAs and plant miRNAs,and effectively solve the non-specific transcription of T7 RNApolymerase,which is of great significance for the study of miRNA-based biological function and the diagnosis of diseases.