Research On MiRNA Detection Based On Aligner-Target-Mediated Cleavage

ObjectiveMicro RNAs（mi RNAs）are a class of short-stranded non-coding RNAs,18-24nucleotides in length,which have crucial roles in various biological processes,such as cell development,proliferation,differentiation and programmed death.A growing body of research indicates that abnormal mi RNA expression contributes to a variety of human diseases,including cancer,neurodegenerative diseases,diabetes,heart disease,kidney disease,liver disease,and altered immune system function.mi RNA has been proposed as a biomarker that can be used for early detection,classification,prognosis,and predictive diagnosis of diseases because abnormal mi RNA expression is closely related to disease onset and progression and can appear in a stable form in body fluids such as blood,saliva,and urine.Therefore,the identification and detection of mi RNAs has significant medicinal implications and developmental potential.MethodIn this paper,we propose an"Identification-Cleavage-Amplification（ICA）"strategy for the detection of different mi RNAs.It combines an aligner-target mediated cleavage with strand displacement amplification（ATMC-SDA）to achieve the"Identification-Cleavage-Amplification"process.First of all,a DNA-aligner（DA）and a DNA-amplicon（DM）can bind together with the help of target mi RNA,forming a T-junction structure.Then,a nicking endonuclease（NEase）,binding on the recognition sequence at the stem part of DA,can make a cleavage on DM,and the cleaved DM（CDM）can serve as an initiator to trigger the strand displacement amplification（SDA）reaction for signal amplification.The specific methods are as follows:to achieve the"identification-cleavage"strategy,this study first designed the corresponding DA and DM sequences according to the sequence of the target mi RNA,constructed the reaction system,and optimized the DA and DM to determine the cleavage product of this method;In order to achieve the"amplification"strategy,this study designed the corresponding forward primer and reverse primer according to the cleaved CDM after the cleavage experiment,and optimized the reaction conditions,such as the amount of NEase,the amount of polymerase,the concentration of Mg²⁺,etc.After that,experiments on sensitivity,specificity,generality and spiked recovery were conducted,and standard curves were established,and finally statistical analysis was performed.ResultThe method has been successfully used for the detection of mi R-155,mi R-21 or let 7a.For the detection of mi R-155,with a linear regression equation of Y=1.111 X+6.074,a correlation coefficient of 0.9987 with a limit of detection（LOD）calculated as 5.6 amol/L.The recovery rates have also been examined,which ranged from 97.7%to 112%with relative standard deviations（RSD）between 1.3%and 2.7%.The linear regression equation for mi R-21 was Y=0.9207 X+2.462 with a correlation coefficient of 0.9996,with an LOD calculated as 3.9 amol/L.The linear regression equation for let 7a was Y=0.7320 X+1.124with a correlation coefficient of 0.9912 and a minimum detection limit of 7.2 amol/L.ConclusionWe herein present an identification-cleavage-amplification（ICA）strategy for highly sensitive and versatile detection of mi RNA.Compared with existing methods,the ATMC-SDA based ICA strategy is very sensitive,with detection limits up to the a M level;furthermore,the method has good specificity,can distinguish single base mismatches;in addition,the method has good versatility.On the one hand,this is because different mi RNAs can be detected by changing only part of the sequence of the 5’end side arm of DA and the 3’end side arm of DM.On the other hand,the design of primers is very simple because the strands involved in amplification,i.e.CDM,can be the same when different mi RNAs are detected The good detection results of ATMC-SDA in real complex samples（e.g.human serum）make it potential to be used in practical assays.Therefore,based on the above advantages,this method can be used for sensitive detection of mi RNAs.