Article

Amplified Self‐Immolative Release of Small Molecules by Spatial Isolation of Reactive Groups on DNA‐Minimal Architectures

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Abstract

Triggering the release of small molecules in response to unique biomarkers is important for applications in drug delivery and biodetection. Due to typically low quantities of biomarker, amplifying release is necessary to gain appreciable responses. Nucleic acids have been used for both their biomarker recognition properties and as stimuli, notably in amplified small molecule release via nucleic acid‐templated catalysis (NATC). The multiple components and reversibility of NATC, however, make it difficult to apply in vivo . Here, we report the first use the hybridization chain reaction (HCR) for the amplified, conditional release of small molecules from standalone nanodevices. We first couple HCR with a DNA‐templated reaction resulting in the amplified, immolative release of small molecules. Moreover, we integrate the HCR components into single nanodevices as DNA tracks and spherical nucleic acids, spatially isolating reactive groups until triggering. Overall, this work translates the amplification of HCR into small molecule release without the use of multiple components, and will aid its application to biosensing, imaging and drug delivery.

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... 17 This strategy of DNA-templated synthesis (DTS) has been used to generate macrocycles, 19 tripeptides, 20 as well as sequence-specific linear and cyclic oligomers. [21][22][23] Processive ''nanowalkers'' 24 and the hybridization chain reaction (HCR) [25][26][27] have both been employed to autonomously generate organic products. These methods of DTS generate molecules based on a coded sequence of inputs: they transfer sequence and spatial information into product structure, generating monodisperse outputs. ...
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Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signalling networks. Synthetic silicon-based circuitry similarly relies on spatial constraints to process information. Here, we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbours, identical DNA hairpins can be reused across circuits. Co-localization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate our approach will motivate using spatial constraints for future molecular control circuit designs.
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Nucleic acid-templated reactions have attracted significant attention for nucleic acid sensing and imaging. The level of signal amplification obtained from templated reactions is a function of the template turnover, wherein the template acts as the catalyst. Herein, we report the application of a pyridinium linker that immolates upon photocatalytic reduction with a ru-thenium complex to yield the fastest nucleic acid tem-plated reaction reported to date. We show that the tem-plated reaction turnover is limited by the duplex dissoci-ation kinetics beyond probes longer than a 6-mer and proceeded fastest for a 5-mer PNA probe. Using a bea-con architecture that masks the catalytic template, we show that this methodology can be used for nucleic acid sensing extending the analyte recognition beyond a 5-mer. The system proceeds with a catalytic efficiency of 105 M-1s-1 and achieves turnover frequency of > 100 h-1.
Article
Theoretical models of localized DNA hybridization reactions on nanoscale substrates indicate potential benefits over conventional DNA hybridization reactions. Recently, a few approaches have been proposed to speed-up DNA hybridization reactions; however, experimental confirmation and quantification of the acceleration factor have been lacking. Here, a system to investigate localized DNA hybridization reactions on a nanoscale substrate is presented. The system consists of six metastable DNA hairpins that are tethered to a long DNA track. The localized DNA hybridization reaction of the proposed system is triggered by a DNA strand which initiates the subsequent self-assembly. Fluorescence kinetics indicates that the half-time completion of a localized DNA hybridization chain reaction is six times faster than the same reaction in the absence of the substrate. The proposed system provides one of the first known quantification of the speed-up of DNA hybridization reactions due to the locality effect.
Article
Nucleic acids are generally regarded as the payload in gene therapy, often requiring a carrier for intracellular delivery. With the recent discovery that spherical nucleic acids enter cells rapidly, we demonstrate that nucleic acids also have the potential to act as a delivery vehicle. Herein, we report an amphiphilic DNA-paclitaxel conjugate, which forms stable micellar nanoparticles in solution. The nucleic acid component acts as both a therapeutic payload for intracellular gene regulation and the delivery vehicle for the drug component. A bioreductively activated, self-immolative disulfide linker is used to tether the drug, allowing free drug to be released upon cell uptake. We found that the DNA-paclitaxel nanostructures enter cells ca. 100 times faster than free DNA, exhibit increased stability against nuclease, and show nearly identical cytotoxicity as free drug. These nanostructures allow one to access a gene target and a drug target using only the payloads themselves, bypassing the need for a co-carrier system.
Article
There is significant interest in developing methods that visualize and detect RNA in live cells. Bioorthogonal template driven tetrazine ligations could be a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens that can be activated via a tetrazine reaction. Herein we report a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near-infrared (NIR) emitting cyanine dye. Vinyl ether caged fluorophores and tetrazine partners are conjugated to high affinity antisense nucleic acid probes, which show highly selective fluorogenic reactivity when annealed to their respective target RNA sequences. A target sequence in the 3' untranslated region (3' UTR) of an expressed mRNA was successfully detected in live cells employing appropriate nucleic acid probes bearing a tetrazine reactive NIR fluorogen. Given the expansion of tetrazine fluorogenic chemistry to NIR dyes, we believe highly selective proximity induced fluorogenic tetrazine reactions could find broad uses in illuminating endogenous biomolecules in living cells.
Article
Molecular machines that assemble polymers in a programmed sequence are fundamental to life. They are also an achievable goal of nanotechnology. Here, we report synthetic molecular machinery made from DNA that controls and records the formation of covalent bonds. We show that an autonomous cascade of DNA hybridization reactions can create oligomers, from building blocks linked by olefin or peptide bonds, with a sequence defined by a reconfigurable molecular program. The system can also be programmed to achieve combinatorial assembly. The sequence of assembly reactions and thus the structure of each oligomer synthesized is recorded in a DNA molecule, which enables this information to be recovered by PCR amplification followed by DNA sequencing.
Article
Designed nanostructures formed by self-assembly of multiple DNA strands suffer from low stability at elevated temperature and under other denaturing conditions. Here, we propose a method to covalent coupling of all DNA strands in such structures by formation of disulfides, which also allows disassembly of the structure under reductive conditions. The dynamic chemistry of disulfides and thiols was applied to cross-link DNA strands with terminal disulfide modifications. The formation of disulfide linked DNA-duplexes consisting of three strands and a more complex DNA double-crossover tile is demonstrated. All the strands in the fully disulfide-linked structures are covalently and geometrically interlocked and it is demonstrated that the structures are stable towards heating and denaturants. Such a reversible system cab be exploited in applications where higher stability of DNA assemblies is needed only temporarily such as, for example, delivery of cargoes to cells by DNA nanostructures.
Article
Selective inhibition of cancer cells remains a challenge in chemotherapy. Here we report the molecular and cellular validation of enzyme-instructed self-assembly (EISA) as a multiple step process for selectively killing cancer cells that overexpress alkaline phosphatases (ALPs). We design and synthesize two kinds of d-tetrapeptide containing one or two phosphotyrosine residues and with the N-terminal capped by a naphthyl group. Upon enzymatic dephosphorylation, these d-tetrapeptides turn into self-assembling molecules to form nanofibers in water. Incubating these d-tetrapeptides with several cancer cell lines and one normal cell line, the unphosphorylated d-tetrapeptides are innocuous to all the cell lines, the mono- and diphosphorylated d-tetrapeptides selectively inhibit the cancer cells, but not the normal cell. The monophosphorylated d-tetrapeptides exhibit more potent inhibitory activity than the diphosphorylated d-tetrapeptides do; the cancer cell lines express higher level of ALPs are more susceptible to inhibition by the phosphorylated d-tetrapeptides; the precursors of d-tetrapeptides that possess higher self-assembling abilities exhibit higher inhibitory activities. These results confirm the important role of enzymatic reaction and self-assembly. Using uncompetitive inhibitors of ALPs and fluorescent d-tetrapeptides, we delineate that the enzyme catalyzed dephosphorylation and the self-assembly steps, together, result in the localization of the nanofibers of d-tetrapeptides for killing the cancer cells. We find that the cell death modality likely associates with the cell type and prove the interactions between nanofibers and the death receptors. This work illustrates a paradigm-shifting and biomimetic approach and contributes useful molecular insights for the development of spatiotemporal defined supramolecular processes/assemblies as potential anticancer therapeutics.
Article
Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.
Article
The ability to probe low-abundance biomolecules or transport high-load drug in targeting cells is essential for biology and theranostics. We develop a novel activatable theranostic approach by using a structure-switching aptamer triggered hybridization chain reaction (HCR) on cell surface, which for the first time creates an aptamer platform enabling real-time activation and amplification for fluorescence imaging and targeting therapy. The aptamer probe is designed not to initiate HCR in its free state but trigger HCR on binding to target cell via structure switching. The HCR not only amplifies fluorescence signals from a fluorescence-quenched probe for activatable tumor imaging, but accumulates high-load prodrugs from a drug-labeled probe and induced its uptake and conversion into cisplatin in cells for selective tumor therapy. In vitro assay shows that this approach affords efficient signal amplification for fluorescence detection of target protein PTK7 with a detection limit of 1 pM. Live cell studies reveals that it provides high-contrast fluorescence imaging and highly sensitive detec-tion of tumor cells, while renders high-efficiency drug delivery into tumor cells via an endocytosis pathway. The results imply the potential of the developed approach as a promising platform for early-stage diagnosis and precise therapy of tumors.
Article
The simultaneous intracellular delivery of multiple types of payloads, such as hydrophobic drugs and nucleic acids, typically requires complex carrier systems. Herein, we demonstrate a self-deliverable form of nucleic acid-drug nanostructure that is comprised almost entirely of payload molecules. Upon light activation, the nanostructure sheds the nucleic acid shell, while the core, which consists of prodrug molecules, disintegrates via an irreversible self-immolative process, releasing free drug molecules and small molecule fragments. We demonstrate that the nanostruc-tures exhibit enhanced stability against DNase I compared with free DNA, and that the model drug (camptothecin) released exhibits similar efficacy as free, unmodified drugs towards cancer cells.
Article
Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ∼3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.
Article
We describe two DNA-templated ligation strategies: native chemical ligation (NCL), and thiol-disulfide exchange. Both systems result in successful ligation in the presence of a DNA template. The stability of the product from the NCL reaction relies on exogenous thiol, while the thiol-disulfide reaction proceeds in a catalyst-free manner.
Article
Die Bemühungen, die chemische Ligation von Oligonukleotiden ohne biochemische Enzyme zu etablieren, haben zu einer Vielzahl an synthetischen Analoga geführt und damit die Oligomer-Ligation um neue Oligonukleotide, Peptide und Hybride, wie PNA, bereichert.1 Wichtigste Anforderungen an potenzielle PCR-freie Diagnosewerkzeuge sind eine schnelle templatierte chemische DNA-Ligation mit hoher Paarungsselektivität, sowie eine empfindliche Detektionsmethode. Hier berichten wir über eine Oligonukleotid-Festphasensynthese von 5′- oder 3′-Mercapto-didesoxynukleotiden. Die chemische Ligation liefert 3′-5′-Disulfid-Bindungen anstelle der 3′-5′-Phosphordiester-Bindung. Unter Verwendung einer Fluoreszenzmethode beschreiben wir eine der schnellsten Ligationsreaktionen mit Halbwertszeiten in der Größenordnung von Sekunden. Durch die Wahl der DNA-Modifikation und der 3′-5′-Orientierung der Aktivierung wird die nicht templatierte Ligation effektiv unterdrückt. Der Temperatureinfluss auf die templatierte Ligation wir aufgezeigt.
Article
Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.
Article
Inspired by biological polymers, sequence-controlled synthetic polymers are highly promising materials that integrate the robustness of synthetic systems with the information-derived activity of biological counterparts. Polymer-biopolymer conjugates are often targeted to achieve this union; however, their synthesis remains challenging. We report a stepwise solid-phase approach for the generation of completely monodisperse and sequence-defined DNA-polymer conjugates using readily available reagents. These polymeric modifications to DNA display self-assembly and encapsulation behavior-as evidenced by HPLC, dynamic light scattering, and fluorescence studies-which is highly dependent on sequence order. The method is general and has the potential to make DNA-polymer conjugates and sequence-defined polymers widely available.
Article
Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA.1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid-phase synthesis of oligonucleotides containing 5'- or 3'-mercapto-dideoxynucleotides and their chemical ligations, yielding 3'-5'-disulfide bonds as a replacement for 3'-5'-phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half-lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3'-5' orientation of the activation site. The influence of temperature on the templated reaction is shown.
Article
The vascular endothelial growth factors (VEGF) and their tyrosine kinase receptors play a pivotal role in angiogenesis and lymphangiogenesis during development and in pathologies such as tumor growth. The VEGFs function as disulfide-linked antiparallel homodimers. The lymphangiogenic factors, VEGF-C and VEGF-D, exist as monomers and dimers, and dimerisation is regulated by a unique unpaired cysteine. In this study, we have characterized the redox state of this unpaired cysteine in a recombinant mature monomeric and dimeric VEGF-C by mass spectrometry. Our findings indicate that the unpaired cysteine regulates dimerisation via thiol/disulfide exchange involving the inter-dimer disulfide bond.
Article
We develop a new homogeneous method for sensitive detection of various biomolecules on the basis of bioluminescence monitoring the released AMP from the target-triggered hybridization chain reaction-mediated ligation. The introduction of hybridization chain reaction not only improves the sensitivity of DNA assay, but also facilitates the sensitive detection of proteins by designing specific aptamer triggers, providing a universally amplified platform for simultaneous detection of different kinds of biomolecules. Importantly, this bioluminescence assay employs the target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. The proposed method has significant advantages of simplicity, high sensitivity, low-cost, and high throughput, and holds a great promise for practical point-of-care applications.
Article
Reactions templated by cellular nucleic acids are attractive for nucleic acid sensing or responsive systems. Herein we report the use of a photocatalyzed reductive cleavage of an immolative linker to unmask a rhodamine fluorophore, and its application to miRNA imaging. The reaction was found to proceed with a very high turnover (>4000) and provided reliable detection down to 5 pM of template by using γ-serine-modified peptide nucleic acid (PNA) probes. The reaction was used for the selective detection of miR-21 in BT474 cells and miR-31 in HeLa cells following irradiation for 30 min. The probes were introduced by using reversible permeation with streptolysin-O (SLO) or a transfection technique.
Article
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.
Article
Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.
Article
Nucleic acid templated reactions have attracted significant attention for nucleic acid sensing. Herein we report a general design which extends the potential of nucleic acid templated reactions to unleash the function of a broad diversity of small molecules such as a transcription factor agonist, a cytotoxic or a fluorophore.
Article
The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. This has enabled the construction of a diverse and rapidly growing set of DNA nanostructures and nanodevices through the programmed hybridization of complementary strands. Although it had initially focused on the self-assembly of static structures, DNA nanotechnology is now also becoming increasingly attractive for engineering systems with interesting dynamic properties. Various devices, including circuits, catalytic amplifiers, autonomous molecular motors and reconfigurable nanostructures, have recently been rationally designed to use DNA strand-displacement reactions, in which two strands with partial or full complementarity hybridize, displacing in the process one or more pre-hybridized strands. This mechanism allows for the kinetic control of reaction pathways. Here, we review DNA strand-displacement-based devices, and look at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.
Article
Multistep synthesis in the laboratory typically requires numerous reaction vessels, each containing a different set of reactants. In contrast, cells are capable of performing highly efficient and selective multistep biosynthesis under mild conditions with all reactants simultaneously present in solution. If the latter approach could be applied in the laboratory, it could improve the ease, speed and efficiency of multistep reaction sequences. Here, we show that a DNA mechanical device--a DNA walker moving along a DNA track--can be used to perform a series of amine acylation reactions in a single solution without any external intervention. The products of these reactions are programmed by the sequence of the DNA track, but they are not related to the structure of DNA. Moreover, they are formed with speeds and overall yields that are significantly greater than those previously achieved by multistep DNA-templated small-molecule synthesis.
Article
This study focuses on the disassembly-behavior of self-immolative pro-fluorescent linkers under physiological conditions and through an enzyme-initiated domino reaction. The targeted linkers are based on para-aminobenzylalcohol (PABA) or hemithioaminal derivatives of para-carboxybenzaldehyde or glyoxilic acid. We found that a fine tuning of the kinetic properties could be obtained through the modulation of the linker structure, giving either a fast signal response or free-adaptable systems suitable for the design of protease-sensitive fluorogenic probes or prodrug systems.