Construction And Application Research Of Toehold Strand Displacement-Assisted Fluorescent Nucleic Acid Sensor

In the past two years,the coronavirus disease（COVID-19）has swept the world and caused millions of deaths.In order to prevent the spread of the epidemic and avoid the large-scale movement of people,early diagnosis also plays a vital role.In the early stage of the outbreak,scientists were committed to proposing various detection methods for COVID-19 detection.In the end,nucleic acid detection with fluorescent PCR for signal amplification stood out because of its rapid and simple advantages and became the most commonly used detection method.More than that,fluorescent nucleic acid sensors are also widely used in the detection of various cancer-related biomarkers that threaten human life.Early cancer diagnosis lays the foundation for cancer treatment.Among them,the toehold-mediated strand displacement reaction（TSDR）takes toehold as a starting point to promote the process of competing hybridization of different nucleic acid molecules with complementary strands to obtain a more stable double-strand,which is widely applied for signal amplification with the characteristics of insusceptibility to external reaction condition.In this paper,three kinds of fluorescent nucleic acid sensors are constructed through a series of TSDRs for the detection of microRNA,messenger RNA（mRNA）and single nucleotide polymorphisms（SNPs）,respectively.The specific research content is summarized as follows:1.Highly sensitive and label-free detection of DILI microRNA biomarker via target recycling and primer exchange reaction amplificaitonsMicro RNAs are closely associated with the development of diseases in life processes,and the sensitive detection of microRNA biomarkers is beneficial to disease diagnosis and treatment at early stage.Herein,by coupling the target sequence recycling with the primer exchange reaction（PER）,a highly sensitive and non-label approach is constructed to detect microRNA-122,the biomarker for drug-induced liver injury.The target sequence cyclically displaces two hairpins on the DNA track via toehold-mediated strand reactions with the assistance of the fuel DNA.The released hairpins further bind the primer to trigger the polymerase-aided PER process for the yield of plenty of G-quadruplex sequences,which then combine with the thioflavin T to drastically enhance its fluorescence for sensing microRNA-122 with a low 49.4 fM detection limit.Highly specific discrimination of the target microRNA-122 can also be realized with the proposed method.Because of the non-label format,high selectivity and sensitivity,such a method can be a convenient and universal means for sensing various biomarkers for disease diagnosis at the early stages.2.Target-triggered autocatalytic sequence recycling for sensitive and simultaneous detection of microRNA and mRNA via multi-donor iFRET signal amplificationSimultaneous monitoring of different biomarker molecules can potentially offer high accuracy for disease diagnosis.In this work,by using a new biological auto-cycling proximity recording（APR）approach and multi-donor-induced fluorescence resonance energy transfer（iFRET）,we establish a multiplexed and sensitive fluorescent method for simultaneous detection of microRNA-155 and osteopontin mRNA for accurate alcoholic liver disease diagnosis.The presence of the two target sequences triggers the APR process via two toehold-mediated strand displacement reactions for the generation of many ds DNAs with the ROX and Cy5 dyes linked to their termini.Subsequent excitation of the SYBR Green I dye intercalated into the ds DNA strands exhibits amplified fluorescence at distinct wavelengths via iFRET for sensitive and simultaneous detection of microRNA-155and osteopontin mRNA.With the synergistic signal amplification by APR and iFRET,our method shows sub-femtomolar detection limits for the two RNA sequences（e.g.,0.5 and0.3 fM for microRNA-155 and osteopontin mRNA,respectively）.In addition,such a method can also realize high selectivity and the detection of the two RNA sequences in diluted serum samples,indicating its potential application for accurate diagnosis of other diseases.3.Invader assay-controlled catalytic self-assembly of multi-DNAzyme for detection of SNPs in K-rasSingle nucleotide polymorphisms（SNPs）are main factors causing differences in human phenotypes,drug resistance and even the development of diseases.Monitoring SNPs is an important part of Human Genetic Project,potentially beneficial to drug design and disease diagnosis.In this work,we established tripartite DNAzyme junctions for efficiently and sensitively detecting SNPs of K-ras gene via an invader assay-controlled catalytic hairpin self-assembly strategy.Unlike wild-type DNA,mutant-type DNA destroys the trinucleotide repeat substrate and causes the incapability of FEN1 to cleave the 5’flap.The supporting strand binds to the probe oligonucleotide from 5’flap,exposing the locked toehold of the K-ras gene,and inducing catalytic hairpin self-assembly to form a tripartite DNAzyme junctions,which cleave the signal probe to keep the quencher（Dabcyl）away from the fluorophore（FAM）with the presence ofMg²⁺,and then restore fluorescence for monitoring SNPs with detection limit as 4.23 fM.Due to its high specificity,efficiency and sensitivity,this strategy can be expected to detect SNPs of other genes.