Zhang Bin, Li Shuo, Guan Yifu, Yuan Ying
Department of Biochemistry and Molecular Biology, School of Life Sciences, China Medical University, Shenyang, Liaoning 110122, China.
Department of Oral-Maxillofacial Surgery and Plastic Surgery, Stomatology Hospital of China Medical University, Shenyang, Liaoning 110002, China.
ACS Omega. 2021 Jan 7;6(2):1516-1522. doi: 10.1021/acsomega.0c05279. eCollection 2021 Jan 19.
The close relationships of miRNAs with human diseases highlight the urgent needs for miRNA detection. However, the accurate detection of a target miRNA in mixed miRNAs of high sequence homology presents a great challenge. Herein, a novel method called target-protection rolling circle amplification (TP-RCA) is proposed for this purpose. The protective probe is designed so that it can form a fully complementary duplex with the target miRNA and can also mismatch duplexes with other nontarget miRNAs. These duplexes are treated with a single strand-specific nuclease. Consequently, only the target miRNA in a perfect-match duplex can resist the cleavage of nuclease, whereas the nontarget miRNAs in mismatched duplexes will be digested completely. The protected target miRNA can be detected using RCA reactions. MicroRNA let-7 family members (let-7a-let-7f) and nuclease CEL I were used as proof-of-concept models to evaluate the feasibility of the TP-RCA method under different experimental conditions. The experimental results show that the TP-RCA method can unambiguously detect the target let-7 species in mixtures of let-7 family members even though they may differ by only a single nucleotide. This TP-RCA method significantly improves the detection specificity of miRNAs.
微小RNA（miRNA）与人类疾病的密切关系凸显了对miRNA检测的迫切需求。然而，在具有高度序列同源性的混合miRNA中准确检测目标miRNA是一项巨大挑战。在此，为此目的提出了一种名为靶标保护滚环扩增（TP-RCA）的新方法。设计保护探针，使其能够与目标miRNA形成完全互补的双链体，也能与其他非目标miRNA形成错配双链体。这些双链体用单链特异性核酸酶处理。因此，只有完美匹配双链体中的目标miRNA能够抵抗核酸酶的切割，而错配双链体中的非目标miRNA将被完全消化。受保护的目标miRNA可通过滚环扩增反应进行检测。使用微小RNA let-7家族成员（let-7a至let-7f）和核酸酶CEL I作为概念验证模型，以评估TP-RCA方法在不同实验条件下的可行性。实验结果表明，即使let-7家族成员混合物中的目标let-7种类可能仅相差一个核苷酸，TP-RCA方法也能明确检测到它们。这种TP-RCA方法显著提高了miRNA检测的特异性。
