Localized Catalytic Hairpin Assembly For MiRNA-21 Highly Sensitive Detection In Living Cell

MicroRNAs(miRNAs)are a class of small and non-protein coding RNA molecules containing 19–24 nucleotides,which play central roles in regulation of gene expressions in various biological processes.lots of studies have suggested a close relationship between the dysregulation of miRNA expressions and human diseases,hence enabling the specific miRNAs highly potential biomarkers for the diagnosis,prognosis as well as treatment of related diseases and even cancers,Thus,in situ detection of miRNAs in live cells will be highly desira ble.Catalytic hairpin assembly(CHA)is an enzyme free and constant-temperature nucleic acid signal amplification technology which is proposed in recent years.It has been used for intracellular RNA detection,but its application in cells face s two problems: on the one hand,it is difficult for the probe to enter the cell autonomously,and on the other hand,the reaction of CHA is slow.In this paper,a localized catalytic hairpin self-assembly technique(LCHA)was designed which was based on CHA.This technique could break through the limitations brought by CHA and designed two methods for detecting intracellular miRNA.The main details are as follows:(1)Based on single-stranded DNA,a DNA backbone was constructed for highly sensitive detection of intracellular miRNAs.A pair of hairpin probes(H1 is a self-quenched hairpin structure by labeling with FAM and BHQ1 at appropriate positions)which have the ability to generate CHA are alternately attached to a DNA backbone,and the DNA backbone can enter the cell autonomously through endocytosis,thereby bringing the hairpin probe into the cell.When the target miRNA is present,the hairpin H1 will be first opened,and the toehold of H2 will hybridize with H1 to replace the target.At this time,FMA and BHQ1 on H1 will move away from each other and the fluorescence will recover.Since the DNA backbone contains many H1 and H2,multiple CHAs are t riggered at the same time.The reaction is LCHA.The detection limit of LCHA is about one order of magnitude higher than that of CHA,and LCHA increases the local concentration of the hairpin,which makes the reaction faster.In addition,the DNA nanoprobe composed of the DNA backbone is capable of detecting dynamic changes of intracellular miRNAs,so it can be used as a simple and effective method for basic research of intracellular miRNAs.(2)In the previous work,our proposed LCHA can speed up the react ion rate,but its false positive signal will also increase.Based on this,we combine FRET with LCHA,which not only accelerates the reaction rate,but also effectively avoids false positive signals.Based on single-stranded DNA,a DNA tetrahedron was constructed for highly sensitive detection of intracellular miRNAs.A pair of hairpin probes that can generate CHA(H1 is a hairpin structure labeled with FAM at an appropriate position,and H2 is a hairpin structure labeled with TAMRA at an appropriate position)attached to a DNA tetrahedron,respectively,and the DNA tetrahedron enters the cell autonomously through endocytosis,thereby bringing the hairpin probe into the cell.When the target miRNA is present,the hairpin H1 is first opened,and the toehold of H2 hybridizes with H1 to replace the target.At this time,H1 and H2 hybridize to form a DNA double strand,and the FAM fluorescent group labeled on H1 and The TAMRA fluorophores labeled on H2 are close to each other,producing a distinct FRET signal,and the displaced target can trigger a new round of CHA to achieve amplification detection.