Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, and Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University , Changsha 410082, China.
Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Health Cancer Center, University of Florida , Gainesville, Florida 32611-7200, United States.
ACS Nano. 2017 Apr 25;11(4):4060-4066. doi: 10.1021/acsnano.7b00725. Epub 2017 Mar 30.
Accurate detection and imaging of tumor-related mRNA in living cells hold great promise for early cancer detection. However, currently, most probes designed to image intracellular mRNA confront intrinsic interferences arising from complex biological matrices and resulting in inevitable false-positive signals. To circumvent this problem, an intracellular DNA nanoprobe, termed DNA tetrahedron nanotweezer (DTNT), was developed to reliably image tumor-related mRNA in living cells based on the FRET (fluorescence resonance energy transfer) "off" to "on" signal readout mode. DTNT was self-assembled from four single-stranded DNAs. In the absence of target mRNA, the respectively labeled donor and acceptor fluorophores are separated, thus inducing low FRET efficiency. However, in the presence of target mRNA, DTNT alters its structure from the open to closed state, thus bringing the dual fluorophores into close proximity for high FRET efficiency. The DTNT exhibited high cellular permeability, fast response and excellent biocompatibility. Moreover, intracellular imaging experiments showed that DTNT could effectively distinguish cancer cells from normal cells and, moreover, distinguish among changes of mRNA expression levels in living cells. The DTNT nanoprobe also exhibits minimal effect of probe concentration, distribution and laser power as other ratiometric probe. More importantly, as a result of the FRET "off" to "on" signal readout mode, the DTNT nanoprobe almost entirely avoids false-positive signals due to intrinsic interferences, such as nuclease digestion, protein binding and thermodynamic fluctuations in complex biological matrices. This design blueprint can be applied to the development of powerful DNA nanomachines for biomedical research and clinical early diagnosis.
准确检测和成像活细胞中的肿瘤相关 mRNA 有望实现癌症的早期检测。然而，目前大多数用于成像细胞内 mRNA 的探针都面临着复杂生物基质所产生的固有干扰，从而导致不可避免的假阳性信号。为了解决这个问题，我们开发了一种称为 DNA 四面体纳米夹（DTNT）的细胞内 DNA 纳米探针，该探针基于 FRET（荧光共振能量转移）“关闭”到“开启”信号读出模式，可可靠地在活细胞中成像肿瘤相关 mRNA。DTNT 由四个单链 DNA 自组装而成。在没有靶 mRNA 的情况下，分别标记的供体和受体荧光团被分离，从而诱导低 FRET 效率。然而，在存在靶 mRNA 的情况下，DTNT 从开环状态转变为闭环状态，从而使双荧光团紧密靠近以实现高 FRET 效率。DTNT 表现出高细胞通透性、快速响应和优异的生物相容性。此外，细胞内成像实验表明，DTNT 可以有效区分癌细胞和正常细胞，并且可以区分活细胞中 mRNA 表达水平的变化。与其他比率探针相比，DTNT 探针的探针浓度、分布和激光功率的影响最小。更重要的是，由于 FRET“关闭”到“开启”信号读出模式，DTNT 探针几乎完全避免了固有干扰（如核酸酶消化、蛋白质结合和复杂生物基质中的热力学波动）引起的假阳性信号。这种设计蓝图可应用于开发用于生物医学研究和临床早期诊断的强大 DNA 纳米机器。
