Department of Chemistry, Liaocheng University, Liaocheng, 252059, PR China; Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100, Jinan, PR China.
Department of Chemistry, Liaocheng University, Liaocheng, 252059, PR China.
Biosens Bioelectron. 2020 Jan 1;147:111788. doi: 10.1016/j.bios.2019.111788. Epub 2019 Oct 17.
The sensitive imaging of telomerase RNA (TR) in living cells is crucial for improved guidance in cancer clinical diagnosis because its expression level is closely related to malignant diseases. The efficient delivery of multiple nucleic acid probes to target cells is critical for nucleic acid-based methods to successfully image low-abundance TR in living cells. While novel nanomaterials enhance delivery efficiency, uncontrolled loading and slow intracellular release remain major challenges for multiple-probe delivery. Here, we designed a facile DNA/RNA nanoflower (NF) to perform the controlled loading of multiple probes and rapid intracellular release based on the "zipper lock-and-key" strategy. First, a long RNA generated by rolling circle transcription acts as both the "smart zipper lock" and the delivery carrier to alternately lock multiple functional DNAs through DNA-RNA base pairing, and the resulting RNA/DNA hybrids self-assemble into packed NFs. The functional DNAs include the fluorescence molecular beacon H for TR recognition, H for hybrid chain reaction (HCR) and DNA-cholesterol for size control. After NF internalization by the cells, the intracellular RNase H acts as the "key" to specifically open the DNA/RNA NFs by cleaving the RNA in the DNA/RNA hybrid, releasing high amounts of H and H in a confined space and thereby facilitating the HCR amplification analysis of cytoplasmic TR. With the addition of a DNA-nuclear localization peptide component in the same NF, nuclear TR can also be sensitively detected. Compared with the regular H/H mixture, the DNA/RNA NFs produced a higher-contrast fluorescence signal. This indicated that the proposed strategy allowed the side arms of H/H to be sealed into the RNA sequence-programmed "zipper lock" by controlled loading, avoiding mutual nonspecific H/H hybridization. In addition, due to the fast kinetics of the RNase endonuclease reaction, the loaded H/H was quickly released. Furthermore, the strategy was successfully used to assay the expression levels of TR in HeLa, HepG2 and HL-7702 cells, demonstrating that this approach holds the potential for the sensitive detection of low-abundance biomarkers in living cells.
端粒酶 RNA（TR）在活细胞中的敏感成像对于癌症临床诊断的改进指导至关重要，因为其表达水平与恶性疾病密切相关。将多个核酸探针有效递送到靶细胞对于基于核酸的方法成功地对活细胞中低丰度 TR 进行成像至关重要。虽然新型纳米材料提高了递送效率，但对于多探针递送，不可控的负载和缓慢的细胞内释放仍然是主要挑战。在这里，我们设计了一种简便的 DNA/RNA 纳米花（NF），基于“拉链锁和钥匙”策略实现了对多个探针的可控负载和快速细胞内释放。首先，通过滚环转录产生的长 RNA 既充当“智能拉链锁”，又充当通过 DNA-RNA 碱基配对将多个功能 DNA 交替锁定的递送载体，所得的 RNA/DNA 杂合体自组装成包装好的 NF。功能 DNA 包括用于 TR 识别的荧光分子信标 H、用于杂交链式反应（HCR）的 H 和用于尺寸控制的 DNA-胆固醇。NF 被细胞内化后，细胞内的 RNase H 作为“钥匙”，通过切割 DNA/RNA 杂合体中的 RNA，特异性地打开 DNA/RNA NF，在有限的空间内释放大量的 H 和 H，从而促进细胞质 TR 的 HCR 扩增分析。在同一 NF 中添加一个 DNA-核定位肽组件，还可以灵敏地检测核 TR。与常规 H/H 混合物相比，DNA/RNA NF 产生了更高对比度的荧光信号。这表明所提出的策略允许通过受控负载将 H/H 的侧臂密封到 RNA 序列编程的“拉链锁”中，避免了 H/H 的非特异性相互杂交。此外，由于 RNase 内切酶反应的快速动力学，负载的 H/H 被快速释放。此外，该策略成功用于测定 HeLa、HepG2 和 HL-7702 细胞中 TR 的表达水平，表明该方法具有在活细胞中灵敏检测低丰度生物标志物的潜力。
