Research On Novel Methods For MiRNA Detection Based On Nucleic Acid Self-assembly Technology

MicroRNAs(miRNAs),with length range from 18 to 25 nucleotides,are a class of single-stranded non-coding RNAs.Considering the importance of miRNA functions in the regulation of gene expression and cellular processes,their potential value as biomarkers in clinical field,it is vital to develop proper detection methods.The traditional methods for detecting miRNA cannot meet the requirement of point-of-care testing(POCT).Therefore,developing rapid,simple,sensitive and high accuracy detection methods for miRNAs are urgently required.Isothermal nucleic acid amplification technology has become the mainstream technology for miRNA detection in the past few years because of its high stability and efficiency.Besides,it eliminates the need for thermal cyclingof polymerase chain reaction.In this study,two new miRNA detection methods of isothermal nucleic acid amplification with miR-200 a were developed,based on phosphorothioated-terminal hairpin formation and self-priming extension(PS-THSP)technology.These two methods presents good sensitivity and sequence-specific discrimination for different target miRNAs.Firstly,the highly simplified DNA detection probe of stem-loop structure was designed for miR-200 a.The efficiency of self-folding has been improved by incorporating phosphorothioates(PS)into the nucleic acid sequences.The PS was used to reduce the thermal stability of double-stranded DNA and promote the folding of hairpin sequences to trigger the amplification reaction.Only when the target miRNA exists,the original stem-loop structure of Pri-C and another primer Pri-D with hairpin sequence can transform into a double-stranded structure of Pri-C / Pri-D hybridization.Under the action of Bst 2.0DNA polymerase with chain replacement activity,the 3’ end of Pri-D was amplified along the Pri-C template chain to produce long fragments of cascading double-stranded DNA products.Then,Pri-C was released for the next catalytic cycle.This design is much simpler and more efficient to detect miRNA at a constant temperature,providing aneasier idea for the development ofnew methods.Taking miR-200 a as the research object,the detection limit of the new method was 350 p M.The miR-200 family sequences(miR-200 a,miR-200 b,miR-200 c and miR-429)and the miR-152 sequences of different families were used to investigate the specificity of this method.In order to improve the detection sensitivity,a new method was established by combining the PS-THSP technique with the catalytic hairpin self-assembly technique(CHA).CHA is a kind of toehold-mediated strand displacementdependent on viscous end‘toehold’.The reaction process follows the principle of base complementary pairing,and the self-assembly of nucleic acids can be achieved at room temperature without the participation of enzymes.CHA has attracted more and more attention due to its excellent non-enzymatic signal amplification characteristics.In this study,two hairpin sequences(H1 and H2)were designed reasonably to realize the process of isothermal nucleic acid amplification.The signal amplification method can be completed in a short time and the specificity is greatly improved.Through catalytic hairpin assembly technique,a large number of H1-H2 double-stranded DNA complexes were formed in the presence of the trace level of target.The stem sequence exposed by H2 could be complementary to the probe of phosphorothioate modification,and then the PS-THSP technique of the above method was started.Therefore,a second cycling signal amplification was achieved to improve detection sensitivity.Under the optimized conditions,the linear range of the detection method was from 5 p M to 250 n M,with the detection limit of 5 p M.Moreover,specificity of this method was investigated and resulted in good performance,includingthe miR-200 family sequences(miR-200 a,miR-200 b,miR-200 c and miR-429)and the miR-152 and let-7a sequences of different families.It provides a possible way for the clinical application of miRNA detection and the further research for other isothermal amplification nucleic acid techniques.