Beijing Advanced Innovation Center for Materials Genome Engineering, University of Science and Technology Beijing, Beijing, 100083, PR China; Beijing Key Laboratory for Bioengineering and Sensing Technology, Research Center for Bioengineering and Sensing Technology, School of Chemistry & Biological Engineering, University of Science & Technology Beijing, Beijing, 100083, PR China.
Beijing Advanced Innovation Center for Materials Genome Engineering, University of Science and Technology Beijing, Beijing, 100083, PR China; Beijing Key Laboratory for Bioengineering and Sensing Technology, Research Center for Bioengineering and Sensing Technology, School of Chemistry & Biological Engineering, University of Science & Technology Beijing, Beijing, 100083, PR China.
Anal Chim Acta. 2019 Oct 31;1078:176-181. doi: 10.1016/j.aca.2019.06.034. Epub 2019 Jun 14.
Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.
细胞内 microRNA (miRNA) 的分析非常有意义，可以深入了解其生理和病理状态，但必须面对基因探针传递和条件释放的关键挑战。在此，我们报告了一种组装的 DNA 迷你六面体 (DMH)，它可以选择性地包装和保护 miRNA 探针，基于对细胞内 miRNA 检测的目标序列识别，实现靶向细胞的特异性传递和释放。简而言之，DMH 是通过合理设计由 6 条单链寡核苷酸链自组装而成的，其中一条含有 AS1411 序列以实现特异性摄取。两个荧光染料标记的识别链通过部分互补杂交插入到两个 DMH 边缘的淬灭基团中。我们发现，这种 DMH 具有很好的生物相容性、良好的跨膜能力，并能够保护基因货物免受酶降解和蛋白质结合的影响。靶介导的竞争性链置换反应引起的荧光恢复允许同时检测两种与癌症相关的细胞内 miRNA，几乎没有假阳性信号，为区分健康正常细胞和癌细胞提供了有力工具。因此，该构建物为克服基因传递、特异性传递和内在干扰抗性方面的挑战开辟了新途径。
