Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead ...to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers, and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research. Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However, this method does not always give an accurate diagnosis and does not allow clinicians to effectively assess the complex molecular alterations that are predictive of cancer progression. As genomics and proteomics do not yet allow a full access to this molecular knowledge, aptamer probes represent one effective and practical avenue toward this goal. One special feature of aptamers is that we can isolate them by selection against cancer cells without prior knowledge of the number and arrangement of proteins on the cellular surface. These probes can identify molecular differences between normal and tumor cells and can discriminate among tumor cells of different classifications, at different disease stages, or from different patients. This Account summarizes our recent efforts to develop aptamers through cell-SELEX for the study of cancer and apply those aptamers in cancer diagnosis and therapy. We first discuss how we select aptamers against live cancer cells. We then describe uses of these aptamers. Aptamers can serve as agents for molecular profiling of specific cancer types. They can also be used to modify therapeutic reagents to develop targeted cancer therapies. Aptamers are also aiding the discovery of new cancer biomarkers through the recognition of membrane protein targets. Importantly, we demonstrate how molecular assemblies can integrate the properties of aptamers and, for example, nanoparticles or microfluidic devices, to improve cancer cell enrichment, detection and therapy.
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.
Exosomes constitute an emerging biomarker for cancer diagnosis because they carry multiple proteins that reflect the origins of parent cells. Assessing exosome surface proteins provides a powerful ...means of identifying a combination of biomarkers for cancer diagnosis. We report a sensor platform that profiles exosome surface proteins in minutes by the naked eye. The sensor consists of a gold nanoparticle (AuNP) complexed with a panel of aptamers. The complexation of aptamers with AuNPs protects the nanoparticles from aggregating in a high‐salt solution. In the presence of exosomes, the non‐specific and weaker binding between aptamers and the AuNP is broken, and the specific and stronger binding between exosome surface protein and the aptamer displaces aptamers from the AuNP surface and results in AuNP aggregation. This aggregation results in a color change and generates patterns for the identification of multiple proteins on the exosome surface.
A coat of many colors: A gold nanoparticle/aptamer biosensor (AuNP/AptX) provides a colorimetric platform for rapid and multiplexed protein analysis of exosomes.
Chemodynamic therapy (CDT) has demonstrated new possibilities for selective and logical cancer intervention by specific manipulation of dysregulated tumorous free radical homeostasis. Current CDT ...methods largely rely on conversion of endogenous hydrogen peroxide (H2O2) into highly toxic hydroxyl radicals via classical Fenton or Haber–Weiss chemistry. However, their anticancer efficacies are greatly limited by the requirement of strong acidity for efficient chemical reactions, insufficient tumorous H2O2, and upregulated antioxidant defense to counteract free radical-caused oxidative damage. Here, we present a new concept whereby bioorthogonal chemistry and prodrug are combined to create a new type of aptamer drug conjugate (ApDC): aptamer-prodrug conjugate (ApPdC) micelle for improved and cancer-targeted CDT. The hydrophobic prodrug bases can not only promote self-assembly of aptamers but also act as free radical generators via bioorthogonal chemistry. In depth mechanistic studies reveal that, unlike traditional CDT systems, ApPdC micelles enable in situ activation and self-cycling generation of toxic C-centered free radicals in cancer cells through cascading bioorthogonal reactions, with no dependence on either H2O2 or pH, yet concurrently with diminished cancerous antioxidation by GSH depletion for a synergistic CDT effect. We expect this work to provide new insights into the design of targeted cancer therapies and studies of free radical-related molecular mechanisms.
Despite bacterial-mediated biotherapies have been widely explored for treating different types of cancer, their implementation has been restricted by low treatment efficacy, due largely to the ...absence of tumor-specific accumulation following administration. Here, the conjugation of aptamers to bacterial surface is described by a simple and cytocompatible amidation procedure, which can significantly promote the localization of bacteria in tumor site after systemic administration. The surface density of aptamers can be easily adjusted by varying feed ratio and the conjugation is able to increase the stability of anchored aptamers. Optimal bacteria conjugated with an average of 2.8 × 10
aptamers per cell present the highest specificity to tumor cells in vitro, separately generating near 2- and 4-times higher accumulation in tumor tissue at 12 and 60 hours compared to unmodified bacteria. In both 4T1 and H22 tumor-bearing mouse models, aptamer-conjugated attenuated Salmonella show enhanced antitumor efficacy, along with highly activated immune responses inside the tumor. This work demonstrates how bacterial behaviors can be tuned by surface conjugation and supports the potential of aptamer-conjugated bacteria for both targeted intratumoral localization and enhanced tumor biotherapy.
Exosomes (Exos) are nanoscale natural vehicles for transporting biomolecules to facilitate cell-to-cell communication, indicating a high potential of them for delivering therapeutics/diagnostics. To ...improve their delivery capacity, a simple, noninvasive, and efficient strategy for functionalizing Exos with effective targeting ligands as well as elucidation of the cellular uptake mechanism of these functionalized Exos was found be to necessary, but remained a challenge. In this work, we used diacyllipid–aptamer conjugates as the targeting ligand to develop an aptamer-functionalized Exos (Apt-Exos) nanoplatform for cell type-specific delivery of molecular therapeutics. The cellular uptake mechanism of Apt-Exos was investigated in details, and distinct behavior was observed in comparison to free Exos. By combining the excellent molecular recognition capability of aptamers and the superiority of Exos as natural vehicles, Apt-Exos can efficiently deliver molecular drugs/fluorophores to target cancer cells, providing a promising delivery platform for cancer theranostics.
The ability to accurately identify and isolate cells is the cornerstone of precise disease diagnosis and therapies. A single-step cell identification method based on logic analysis of multiple ...surface markers will have unique advantages because of its accuracy and efficacy. Herein, using multiple DNA aptamers for cancer biomarker recognition and associative toehold activation for signal integration and amplification as two molecular keys, we have successfully operated a cell-surface device that can perform AND Boolean logic analysis of multiple biomarkers and precisely label the target cell subtype in large populations of similar cells via the presence or absence of different biomarkers. Our approach can achieve single-step cancer cell identification and isolation with excellent sensitivity and accuracy and thus will have broad applications in biological science, biomedical engineering, and personalized medicine.
Nanotechnology has allowed the construction of various nanostructures for applications, including biomedicine. However, a simple target-specific, economical, and biocompatible drug delivery platform ...with high maximum tolerated doses is still in demand. Here, we report aptamer-tethered DNA nanotrains (aptNTrs) as carriers for targeted drug transport in cancer therapy. Long aptNTrs were self-assembled from only two short DNA upon initiation by modified aptamers, which worked like locomotives guiding nanotrains toward target cancer cells. Meanwhile, tandem “boxcars” served as carriers with high payload capacity of drugs that were transported to target cells and induced selective cytotoxicity. aptNTrs enhanced maximum tolerated dose in nontarget cells. Potent antitumor efficacy and reduced side effects of drugs delivered by biocompatible aptNTrs were demonstrated in a mouse xenograft tumor model. Moreover, fluorophores on nanotrains and drug fluorescence dequenching upon release allowed intracellular signaling of nanotrains and drugs. These results make aptNTrs a promising targeted drug transport platform for cancer theranostics.
Hydrogels are crosslinked hydrophilic polymers that can absorb a large amount of water. By their hydrophilic, biocompatible and highly tunable nature, hydrogels can be tailored for applications in ...bioanalysis and biomedicine. Of particular interest are DNA-based hydrogels owing to the unique features of nucleic acids. Since the discovery of the DNA double helical structure, interest in DNA has expanded beyond its genetic role to applications in nanotechnology and materials science. In particular, DNA-based hydrogels present such remarkable features as stability, flexibility, precise programmability, stimuli-responsive DNA conformations, facile synthesis and modification. Moreover, functional nucleic acids (FNAs) have allowed the construction of hydrogels based on aptamers, DNAzymes, i-motif nanostructures, siRNAs and CpG oligodeoxynucleotides to provide additional molecular recognition, catalytic activities and therapeutic potential, making them key players in biological analysis and biomedical applications. To date, a variety of applications have been demonstrated with FNA-based hydrogels, including biosensing, environmental analysis, controlled drug release, cell adhesion and targeted cancer therapy. In this review, we focus on advances in the development of FNA-based hydrogels, which have fully incorporated both the unique features of FNAs and DNA-based hydrogels. We first introduce different strategies for constructing DNA-based hydrogels. Subsequently, various types of FNAs and the most recent developments of FNA-based hydrogels for bioanalytical and biomedical applications are described with some selected examples. Finally, the review provides an insight into the remaining challenges and future perspectives of FNA-based hydrogels.
We survey advances in bioanalytical and biomedical applications of functional nucleic acid-based hydrogels in this review.
Among the vast number of recognition molecules, DNA aptamers generated from cell-SELEX exhibit unique properties for identifying cell membrane biomarkers, in particular protein receptors on cancer ...cells. To integrate all recognition and computing modules within a single structure, a three-dimensional (3D) DNA-based logic gate nanomachine was constructed to target overexpressed cancer cell biomarkers with bispecific recognition. Thus, when the Boolean operator “AND” returns a true value, it is followed by an “ON” signal when the specific cell type is presented. Compared with freely dispersed double-stranded DNA (dsDNA)-based molecular circuits, this 3D DNA nanostructure, termed DNA-logic gate triangular prism (TP), showed better identification performance, enabling, in turn, better molecular targeting and fabrication of recognition nanorobotics.
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