Construction And Application Of High-specificity MicroRNAs Detection System Based On CEL I And RCA

Objective: To build up a low-cost,high-specificity and high-sensitivity nucleic acid detection technology based on the digestion ability of CEL I and the technical characteristics of RCA,which can meet the technical requirements of POCT and evaluate its application value in mi RNA detection.Methods: In this study,a series of 30 bp double-strands oligonucleotides containing different kinds of single-base mismatches were used to investigate the ability and characteristics of CEL I to recognize and hydrolyze DNA double-strands containing single-base mismatches,and attempted to construct a CEL I catalyzed probe-primer protection system.In RCA system,the circular template that could be hybridized with primers specifically was designed and purified by nucleic acid exonuclease after circulization reaction to remove the ligation sequence and uncirculized template sequence.Single-stranded oligonucleotide let-7a DNA and double-stranded oligonucleotide of let-7a DNA/clet-7a were used to trigger the RCA reaction as primers seperatly.Compared and investigated the RCA outcomes triggered by ss-primer and ds-primer.single-stranded to establishe a RCA technology based on the double-stranded primers.Then based on these two systems,a CEL I-RCA system was established to detect the let-7a DNA under the interference nucleotides let-7e DNA which has only one base difference from let-7a DNA.And the detection specificity was evaluated by the resluts.Furthermore,the quantitative detection performance of the system was evaluated by a relative quantitative blind test on target nucleic acid according to the reference system of target nucleic acid with different concentration.In order to optimize the fluorescence performance and stability of RCA system,the influence of circular templates with different nucleotide composition on the fluorescence results of RCA was further studied.A series of oligonucleotide chains with different nucleotide combinations were designed and circularized to be the circular templates and their RCA fluorescence curves indicated by SYBR Green II(SG II)was compared.The nucleotide preference of SG II was investigated by using synthetic oligonucleotide chains composed of different nucleotides.By comparing the fluorescence curves of SG II and SYBR Green I(SG I)fluorescent in RCA reaction,the potential secondary structure of the RCA products from the circular templates composed of different nucleotides was analyzed,and the sequence of the circular template of RCA was optimized.The ability of CEL I to hydrolyze DNA/RNA double-strands containing mismatched bases was investigated by synthetic let-7family micro RNAs(let-7a-let-7f)and their c DNAs,and the reaction system was optimized so that they could be used for direct detection of micro RNAs.Finally,by comparing the detection results of let-7 family micro RNAs with that of Taqman probe RT-q PCR,the differences in detection specificity and resolution between the two methods were evaluated,and the application potential of CEL-based RCA high specificity nucleic acid detection system in POC detection equipment was evaluated.Results: CEL I could hydrolyze ds DNA containing mismatched bases under specific conditions(concentration,temperature,reaction time),and retain target nucleic acid chains that are completely complementary to DNA probes.Complementary double-strands oligonucleotides can be used as primers for RCA reaction.Compared with single-strands oligonucleotides as primers,their amplification curves have the same concentration dependence.The CEL I-RCA highly specific nucleic acid detection system constructed by the combination of the two methods can specifically detect DNA oligonucleotide chains with only one base difference.And the target DNA oligonucleotide chain can be quantitatively detected by reference to standard concentration samples.The fluorescence results of the RCA indicated by SG II showed that SG II only produces strong fluorescence signals with guanine nucleotides,but weak fluorescence signals with other three nucleotides.Circular templates containing large amounts of AC nucleotides can show a linear increase in fluorescence signal in RCA indicated by SG II.While the circular templates containing large amounts of TC nucleotides exhibit linearly increased fluorescence signals in RCA indicated by SG I.AG-rich nucleic acid chains have potential double-chain-like structures,which can produce strong fluorescence with SG I.Further study on the ability of CEL I-RCA system to detect micro RNAs directly showed that CEL I could hydrolyze DNA/RNA double strands containing mismatched bases formed by the hybridization of let-7 family micro RNAs with DNA probes.And the non-specific amplification caused by CEL I can be eliminated by inactivating CEL I at high temperature.The detection results for let-7 family mi RNAs showed that CEL I-RCA nucleic acid detection system could detect let-7 family members with high resolution,and its ability of eliminating non-specific amplification caused by interference mi RNAs was better than that of Taqman probe RT-PCR.Conclusion:(1)CEL I can not only hydrolyze single-strand nucleic acid,but also completely hydrolyze double-strands DNA/DNA or DNA/RNA oligonucleic acid containing single base mismatch.And the specificity of nucleic acid detection can be improved by the characteristic of CEL I.(2)Completely complementary double-strands oligonucleotides can also be used as RCA primers,and their amplification curves have a good concentration dependence.(3)RCA fluorescent indicator SYBR Green II has obvious nucleotide preference and only produces strong fluorescent signals with guanine.(4)When SYBR Green II was used as the fluorescence indicator,the RCA template of AC-rich nucleotide combination had more stable linear fluorescence curve.(5)CEL I-RCA high specificity nucleic acid detection system can distinguish nucleic acid chains with only one base difference.This method not only can detect nucleic acid samples quantitatively,but also has better specificity than RT-PCR.