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.
DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.
The inner region of solid tumors is found to be high-pressure, hypoxic, and immunosuppressive, providing a breeding ground for tumor aggressiveness and metastasis. While intratumoral accumulation of nanomedicines combined with immunomodulation would significantly enhance therapeutic efficacy, such potential is challenged by the compressed environment and distinct heterogeneity of the tumor bulk. By using an apoptotic body (AB) as the carrier, we develop an effective and universal intratumoral nanomedicine delivery system for the long-lasting remission of tumors. Our results show that the AB-encapsulated nanomedicine (using CpG immunoadjuvant-modified gold–silver nanorods as a model), after intravenous injection, can be specifically phagocytosed by inflammatory Ly-6C+ monocytes, which then actively infiltrate the tumor center via their natural tumor-homing tendency. With the integration of AB-facilitated intratumoral accumulation, the nanorod-based photothermal effect, and CpG-promoted immunostimulation, this cell-mediated delivery system can not only efficiently ablate primary tumors but also elicit a potent immunity to prevent tumors from metastasizing and recurring.
Polymeric micelles have received increased attention in the field of pharmaceutical exploitation. However, supra-100-nm micelles, suitable for the EPR effect, cannot penetrate through the dense collagen matrix in solid tumor tissues, thus decreasing the efficacy of anticancer agents. In this work, amphiphilic nucleic acid polymers with tunable hydrophobicity were designed, and size-tunable nucleic acid assemblies were developed to resolve the conflict between EPR effect and spatially uniform penetration ability.
Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six‐letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six‐letter DNA library to selectively bind liver cancer cells; and 2) in a six‐letter self‐assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer‐nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer‐targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer.
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