State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University , Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province , Changsha 410082 , China.
Anal Chem. 2019 Oct 1;91(19):12538-12545. doi: 10.1021/acs.analchem.9b03420. Epub 2019 Sep 12.
The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast but also arrays with plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0-7.0) with a transition midpoint (pH) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells.
高效且精确地将反义寡核苷酸（ASO）递送到靶细胞对于基因沉默非常有价值。然而，递送系统的特异性和包装能力仍然具有挑战性。在这里，我们设计了一种 i -motif 形成引发的原位双足杂交链式反应（pH-Apt-BiHCR）扩增策略，用于特定靶细胞成像和增强 ASO 的基因传递。作为概念验证，使用修饰有锁核酸（LNA）的 8-nt ASO，该 ASO 与 microRNA21（miR-21）的种子区域互补，用于基因沉默研究。得益于发夹包含 i-motif 的设计，基于适体的特异性识别，成功地在 MCF-7 细胞表面上实现了 pH-Apt-BiHCR 的刺激响应组装。利用该策略，pH-Apt-BiHCR 不仅包含重复的荧光共振能量转移（FRET）单元，可用于具有高对比度的可激活肿瘤成像，而且还排列有大量的 LNA ASO 作为干扰分子以抑制癌细胞。体外实验表明，该策略在缓冲液中具有出色的响应能力，在狭窄的 pH 范围内（6.0-7.0），转变中点（pH）为 6.44±0.06。此外，活细胞研究表明，它实现了靶细胞的特异性可激活成像，而排列有 pH-Apt-BiHCR 的 ASO 则通过内吞作用途径实现了更好的内化，并增强了对 MCF-7 细胞的基因沉默作用，与单独使用单个 ASO 相比。我们相信，这种设计将激发用于癌症细胞早期诊断和治疗的新型探针的开发。
