Photoelectrochemical Assay Of MicroRNA And Its Application Investigation

MicroRNAs(MiRNAs)are a kind of short endogenous nonprotein-coding RNA.They distribute boardly in the organisms of plants and animals and function as factors to regulate the expression of gene after the transcription,which mainly via targeting mRNAs to induce the degradation of mRNAs or inhibit the translation of mRNAs.More and more evidence has confirmed that abnormal expression of miRNA is directly or indirectly associated with many diseases,including every kind of cancers.However,the profiling of miRNA expression is very challenging because of their small size,highly similar sequence among family members,and low expression in cells,which also makes it different from detecting other RNAs,such as messanger RNA,and ribosomal RNA etc.The current widely used methods for miRNA profiling are Northern blotting,microarrays,and real-time PCR.Northern blotting is a timeand sample-consuming and semiquantitative detection method with low sensitivity and throughput.In addition,it needs elaborate separation and enrichment processes,and it is very fragile towards RNase contaminations.Analysis techniques based on microarray cost much on producing and detecting.The sample volume for every chip is every low,which significantly lower the sensitivity.The real-time PCR(RT-PCR)approach shows excellent sensitivity,but it requires complex and tedious steps for miRNAs isolation and purification.It also needs to reverse transcription to cDNA prior to the amplification step.Additionally,the apparatus for RT-PCR is very expensive.Thus,developing a fast,specific,sensitive,and convenient method for miRNA profiling analysis is urgently needed and significantly important.(1)Based on the isothermal cycle hybridization amplification of microRNA-21,we developed a selective and sensitive photoelectrochemical assay for microRNA-21 that originated from the specific cleavage activity of duplexspecific nuclease(DSN)toward the DNA strand in the DNA-RNA double helix and in situ producing electron donor of ascorbic acid catalyzed by ALP.After target microRNA-21 hybridized with the complementary biotin-functionalized DNA probe,the DNA probe strand in the DNA-RNA hybridized helix would be cleaved by DSN and microRNA-21 could be released back to the solution.Then the free microRNA could be further hybridized with the remaining single-strand DNA probe on the electrode again;thus,the isothermal cycle hybridization amplification would be formed.As a result,some avidin-alkaline phosphatase conjugate could be captured by the remaining biotin on the electrode.The obvious photocurrent change between a control electrode and the DSN disgested electrode achieved the label-free profiling of microRNA-21 expression with the linear range from 1 to 500 fM.The fabricated biosensor showed high detection selectivity even for one-base mismatched sequence RNA.And we successfully profiled the change of expression level of microRNA-21 in chicken fibroblast cells infected with subgroup J avian leukosis virus.(2)miRNAs are intriguing these days due to its close relationship with human malignancies.To explore biosensors for sensitive and specific detecting miRNAs in human serum blood or organism is necessary and urgent.Taking advantage of the special ligase activity of T4 RNA ligase 2 and exodeoxyribonuclease activity of T7 exonuclease,we develop a two-stage cyclic enzymatic amplification method(CEAM)for the assay of miRNA-21 in human serum blood.Only the specific sequence of mi RNA-21 can initial the first step ligation reaction and then the first stage CEAM.The process of the second-stage CEAM is depending on the product of the first-stage CEAM.After the two-stage CEAM,the concentration of miRNA-21 will be sensitively transcribed to the final amplified signal.The two-stage CEAM achieved a low detection limit of 0.36 fM.This assay presents excellent specificity with discriminating only a single-base mismatched miRNA sequence.Furthermore,this work can also be applied to detect the increased expression level of miRNA-21 in the serum blood of gastric cancer patients,which will be helpful to unveiling the pathway of miRNA-21 involved in the gastric cancer.(3)MicroRNAs play a crucial role in regulating plant growth,such as involving in the pathways of phytohormons signaling,which function in broad range of important aspects in many stages of plant development and evolution.To figure out the components part of microRNA in phytohormone signaling network,a sensitive photoelectrochemical biosensor was developed to detect miRNA-319 a specifically using CuO-CuWO4 as photoactive material and in situ generation electron donor technique.Taking the advantage of rolling circle amplification,nicking endonuclease triggered exponential amplification of target molecule,specific interaction between phosphate group and phos-tag and between biotin and avidin,the developed strategy can greatly amplify the detection signal only using trace amount of target miRNA-319 a.The wide linear range was obtained from 0.1 nM to 1 fM and the detection limit was estimated to be 0.13 fM(S/N=3).The developed method also showed good detection specificity.The effect of five phytohormones on the expression level of miRNA-319 a in rice leaves was also investigated.This will be a basal principle of investigating the signaling network of phytohormones.