Department of Obstetrics and Gynecology, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266003, People's Republic of China.
College of Chemistry and Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China.
J Nanobiotechnology. 2021 Sep 26;19(1):288. doi: 10.1186/s12951-021-01040-x.
Small interfering RNA (siRNA) has emerged as a kind of promising therapeutic agents for cancer therapy. However, the off-target effect and degradation are the main challenges for siRNAs delivery. Herein, an enzyme-free DNA amplification strategy initiated by a specific endogenous microRNA has been developed for in situ generation of siRNAs with enhanced gene therapy effect on cervical carcinoma.
This strategy contains three DNA hairpins (H1, H2/PS and H3) which can be triggered by microRNA-21 (miR-21) for self-assembly of DNA nanowheels (DNWs). Notably, this system is consistent with the operation of a DNA logic circuitry containing cascaded "AND" gates with feedback mechanism. Accordingly, a versatile biosensing and bioimaging platform is fabricated for sensitive and specific analysis of miR-21 in HeLa cells via fluorescence resonance energy transfer (FRET). Meanwhile, since the vascular endothelial growth factor (VEGF) antisense and sense sequences are encoded in hairpin reactants, the performance of this DNA circuit leads to in situ assembly of VEGF siRNAs in DNWs, which can be specifically recognized and cleaved by Dicer for gene therapy of cervical carcinoma.
The proposed isothermal amplification approach exhibits high sensitivity for miR-21 with a detection limit of 0.25 pM and indicates excellent specificity to discriminate target miR-21 from the single-base mismatched sequence. Furthermore, this strategy achieves accurate and sensitive imaging analysis of the expression and distribution of miR-21 in different living cells. To note, compared to naked siRNAs alone, in situ siRNA generation shows a significantly enhanced gene silencing and anti-tumor effect due to the high reaction efficiency of DNA circuit and improved delivery stability of siRNAs.
The endogenous miRNA-activated DNA circuit provides an exciting opportunity to construct a general nanoplatform for precise cancer diagnosis and efficient gene therapy, which has an important significance in clinical translation.
小干扰 RNA（siRNA）已成为癌症治疗的一种有前途的治疗药物。然而，siRNA 递呈的脱靶效应和降解是主要挑战。在此，我们开发了一种无酶 DNA 扩增策略，该策略由特定的内源性 microRNA 引发，用于原位生成 siRNA，从而增强宫颈癌的基因治疗效果。
该策略包含三个 DNA 发夹（H1、H2/PS 和 H3），它们可以被 microRNA-21（miR-21）触发，以自组装 DNA 纳米轮（DNWs）。值得注意的是，该系统与包含级联“与”门和反馈机制的 DNA 逻辑电路的操作一致。因此，构建了一种多功能生物传感和生物成像平台，通过荧光共振能量转移（FRET）对 HeLa 细胞中的 miR-21 进行灵敏和特异性分析。同时，由于血管内皮生长因子（VEGF）反义序列和正义序列编码在发夹反应物中，该 DNA 电路的性能导致 VEGF siRNA 在 DNWs 中的原位组装，这些 siRNA 可以被 Dicer 特异性识别和切割，用于宫颈癌的基因治疗。
所提出的等温扩增方法对 miR-21 具有高灵敏度，检测限为 0.25 pM，并具有优异的特异性，可从单碱基错配序列中区分靶标 miR-21。此外，该策略可实现不同活细胞中 miR-21 表达和分布的精确和灵敏成像分析。值得注意的是，与单独的裸 siRNA 相比，由于 DNA 电路的高反应效率和 siRNA 递呈稳定性的提高，原位 siRNA 生成显示出显著增强的基因沉默和抗肿瘤效果。
内源性 miRNA 激活的 DNA 电路为构建用于精确癌症诊断和高效基因治疗的通用纳米平台提供了一个令人兴奋的机会，这在临床转化中具有重要意义。
