Specific discrimination and universal signal amplification for RNA detection by coupling toehold exchange with RCA through nucleolytic conversion of a structure-switched hairpin probe

• Published in: Analytica chimica acta 2019-08, Vol.1068, p.96-103
• Main Authors: Yu, Wen, Li, Juqiong, Zuo, Chen, Tao, Yiyi, Bai, Shulian, Li, Junlong, Zhang, Zhang, Xie, Guoming
• Format: Article
• Language: English
• Subjects: Amplification; Circularity; Conversion; Exchanging; Fluorescence biosensing; Homology; Lysates; miRNA; miRNA detection; Recognition; Ribonucleic acid; RNA; Rolling circle amplification; Selectivity; Sequences; Strand displacement
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2019.04.016

Herein, we combined toehold exchange with ligation-free rolling circle amplification (RCA) by programming nucleolytic conversion of hairpin probe into sensors, allowed for both high specific recognition and universal signal amplification for RNA detection. The rational engineered HP ensured highly specific recognition based on toehold exchange and allowed the pre-formed circular template for RCA to be shared for different RNAs detection. Generally, detecting different RNA requires different circular template for signal amplification. In this paper, the circular template for RCA was independent of the sequences of the target, so the signal amplification system was an universal one for different RNAs detection. Taking miRNA let-7d as a model target, this method showed a wide linear range from 1 fM to 1 nM with a detection limit of 0.46 fM and exhibited a remarkable selectivity even in distinguishing homologous miRNAs with 1-nt or 2-nt difference. To evaluate the potential of the method, it was applied to analysis the let-7d concentration in human serum, total RNA, and cell lysates. In conclusion, we believe this method exhibits potential for both specific discrimination and universal signal amplification for RNA analysis in complex matrices. Here, we combine toehold exchange with ligation-free rolling circle amplification (RCA) by programming nucleolytic conversion of hairpin probe (HP) into sensors, allows for both high specific recognition and universal signal amplification for RNA detection. This method shows high sensitivity with a detection limit of 0.46 fM and exhibits a remarkable selectivity even in distinguishing homologous miRNAs with 1-nt or 2-nt difference. [Display omitted] •Specific and universal RNA detection was achieved by coupling toehold exchange with ligation-free RCA.•This method shows high sensitivity for miRNA let7d detection with a detection limit of 0.46 fM.•This method exhibits a remarkable selectivity even in distinguishing homologous miRNAs with 1-nt or 2-nt difference.