Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, P R China.
Department of pharmacy, University-Town Hospital of Chongqing Medical University, Chongqing 401331, P R China.
Theranostics. 2018 Mar 27;8(9):2424-2434. doi: 10.7150/thno.23852. eCollection 2018.
While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare. In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield. Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2). This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.
虽然对 microRNA 的检测无论是否有信号放大都具有重要意义，但具有高特异性的用于细胞特异性 RNA 检测的纳米传感器却很少见。在本研究中，将具有特定功能的四面体 DNA 纳米结构（TDN）与具有荧光猝灭效应和大表面积的金纳米粒子（Au-NP）相结合，制造了基于荧光共振能量转移的纳米传感器（Au-TDNN）。miR-21（靶标）的存在可以将 Au-TDNN 上的荧光染料标记的检测探针与 Au-NP 分离，从而分离供体和受体，从而诱导强烈的荧光信号。通过合理设计核酸链置换反应，使其根据热力学参数自发发生 ΔG≈0，从而实现了对区分点突变靶标的高特异性；在这种情况下，碱基错配引起的微小热力学变化对杂交产率有显著影响。对三种单碱基改变的目标、let-7d 和 miR-200b 的化学合成 DNA 进行了测试。预期在 Au-NP 上的检测探针产生了 15.4 的区分因子（DF），用于近端单碱基错配。作为对照组，普通检测探针在 Au-NP 上产生的 DF 仅达到 2.4。还使用肝癌细胞（HepG2）证实了所提出策略的可行性。这种改进的纳米传感器为在活细胞中特异性和易于检测 microRNA 开辟了新途径。
