Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
Anal Chem. 2018 Feb 6;90(3):1502-1505. doi: 10.1021/acs.analchem.7b04848. Epub 2018 Jan 5.
Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution, or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables one to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.
杂交链式反应（HCR）电路是监测低丰度 mRNA 的有价值方法，而当前的 HCR 仍然存在扩增效率有限、定位分辨率受损或设计复杂等问题。我们报告了一种新型的分支 HCR（bHCR）电路，用于在活细胞中对 mRNA 进行高效信号放大成像。bHCR 可以通过分层耦合两个 HCR 电路来实现，其中次级 HCR 电路的两个分裂起始片段被整合到用于初级 HCR 电路的探针中。bHCR 电路可以从单个靶标起始物生成超支化组装，从而有可能在活细胞中定位单个靶标分子。体外实验表明，bHCR 在单碱基错配鉴别中具有非常高的扩增效率和特异性，检测限为 500 fM。活细胞研究表明，bHCR 在活细胞中显示出强烈的荧光斑点，表明 mRNA 定位，对比度得到改善。bHCR 方法可为细胞生物学和诊断学中的低丰度生物标志物检测和成像提供一个有用的平台。
