Application Of Isothermal Exponential Amplification Reaction(EXPAR)on Detection Of MRNA Mutation And Research On Its Non-specific Amplification And Amplification Efficiency

Messenger RNA(mRNA)plays a key role in gene expression as a bridge to translate the genetic information from DNA into functional proteins.It has reported that the site-specific mutations in mRNA can directly affect the expression of functional proteins,which is closely related to the occurrence and development of various diseases,including cancer.So,the sensitive detection of tumor-related mutant mRNAs might be more reliable and valuable in early clinical diagnosis,compared with circulating DNA mutation analysis.However,clinically significant mRNAs with site-specific mutations are typically present in extremely low abundance in heterogeneous samples.Therefore,the accurate detection of trace mRNA mutants in a large pool of normal wild sequences necessitates the development of highly sensitive and highly specific methods.Recently,isothermal exponential amplification reaction(EXPAR)is widely used for the quantitative detection of nucleic acids due to its simplely and high sensitivity.In this paper,we propose a simple but versatile method for mutRNA analysis with single-nucleotide resolution and without the requirement of reverse transcription.This new strategy integrates a new site-specific RNase H cleavage mechanism for highly selective mutation recognition with EXPAR for highly efficient signal amplification(RNase H-EXPAR),resulting in both high sensitivity and specificity for mutRNA analysis.We well-designed a chimeric oligonucleotide probe(abbreviated as cmDNA)with four 2'-O-methyl-modified nucleotides,which can selectively guide RNase H for disconnection at the exact mutation site in the mutRNA,and releasing a newly exposed 3'-OH end fragment.Then the newly exposed 3'-OH end fragment of mutRNA will act as the trigger of EXPAR to initiate the subsequent efficient amplification reaction.By contrast,the cmDNA will not hybridize with wild-type RNA(wtRNA)because the single-base mismatch will lead to decrease the melting temperature of cmDNA/wtRNA heteroduplex.So,at the optimal reaction temperature,the wtRNA will not be hydrolyze by RNase H,which will not trigger the subsequent EXPAR.The RNase H-EXPAR strategy for detecting RNA mutation not only leads to high detection sensitivity that as low as 1 fM mutRNA can be detected,but also effectively avoids the potential false positive interference arising from wtRNA,which allows for the accurate discrimination of 0.1%mutant RNA in a high background of wild RNA sequences.Therefore,we believe that this simple but high specificity and sensitivity approach may be of great significance for studying cancer-related mRNA mutation.EXPAR technology is the promising strategy for nucleic acids amplification because of its facile manipulation and ultrahigh amplification efficiency performance.Although EXPAR technique has many advantages,the performance of EXPAR to detect nucleic acids are seriously hampered due to the two inherent defects of non-specific amplification and invalid trigger.Herein,we designed a novel asymmetric template(As-ddC template)with 3'-terminal blocked by the dideoxycytidine group blocker and added six consecutive 2'-O-methyl-modified nucleotides in the 3'-region(3'-LT).Using specific dideoxycytidine group blocker instead of ordinary phosphate group blocker increase the blocking efficiency to reduce the non-specific amplification.Meanwhile,the six consecutive 2'-O-methyl-modified nucleotides are added in 3'-region of the As-ddC template,making trigger(X)more inclined to hybrid with the 3'-LT to improve the amplification efficiency.We applied our findings on three kinds of miRNAs(mir-122,mir-125b,mir-24)with large differences in detection limits by EXPAR detection.As a result,the proposed approache significantly improves the sensitivity and inhibits the non-specific amplification.Compared with the normal EXPAR strategy,the EXPAR detection limit decreases 3?4 orders of magnitude by our newly designed As-ddC template and the concentrations as low as amol/L targets oligonucleotide could be easily traced.Therefore,we believe that this simple strategy could not only effectively improve the inherent defects of EXPAR but also provide useful references for other isothermal amplification to improve their sensitivity and practicability performances.