Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
Talanta. 2019 Jan 15;192:175-181. doi: 10.1016/j.talanta.2018.07.090. Epub 2018 Jul 30.
Herein, DNA nanostructures were prepared via a palindromic padlock probe-based rolling circle amplification (called P-RCA) and then employed to implement the sensitive and specific detection of let-7a miRNA extracted from cancer cells without chemical modification. The presence of target let-7a miRNA as a polymerization primer can trigger the P-RCA process, generating a long tandemly repetitive DNA strand. The resulting products can fold into nanostructures via self-hybridization of palindromic regions and possess numerous double-stranded fragments. In this case, the strong fluorescent signal is detected upon exposure to SYBR Green I. As a result, in homogeneous solution, target miRNA can be detected down to 6.4 pM with a wide dynamic range. A high specificity was demonstrated by the excellent discrimination between let-7 miRNA family members, while the applicability of this sensing system in complex biological environments was confirmed by the analysis of target miRNAs extracted from HeLa cells. It should be noted that increasing numbers of palindromic fragments in padlock probe further increases signal amplification efficiency. The experimental results indicate that the newly proposed P-RCA DNA nanostructures have potential to become a promising analytical platform in biomedical research and clinical diagnosis for the miRNA detection with high sensitivity and good specificity.
在此，通过基于发夹环探针的滚环扩增（称为 P-RCA）制备了 DNA 纳米结构，然后无需化学修饰即可从癌细胞中提取的 let-7a miRNA 来实现灵敏和特异的检测。目标 let-7a miRNA 作为聚合引物的存在可以触发 P-RCA 过程，生成长串联重复 DNA 链。所得产物可以通过回文区域的自杂交折叠成纳米结构，并具有许多双链片段。在这种情况下，暴露于 SYBR Green I 时会检测到强荧光信号。因此，在均相溶液中，可以检测到低至 6.4 pM 的靶 miRNA，具有较宽的动态范围。通过出色地区分 let-7 miRNA 家族成员，证明了该传感系统具有很高的特异性，同时通过分析从 HeLa 细胞中提取的靶 miRNA 证实了该传感系统在复杂生物环境中的适用性。值得注意的是，发夹环探针中回文片段数量的增加进一步提高了信号放大效率。实验结果表明，新提出的 P-RCA DNA 纳米结构具有成为生物医学研究和临床诊断中 miRNA 检测的有前途的分析平台的潜力，具有高灵敏度和良好的特异性。
