Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been ...developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence
in situ
hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.
This review provides a comprehensive overview of the fundamental principles, analysis techniques, and application fields of hybridization chain reaction and its development status.
DNA has become a promising material to construct high-order structures and molecular devices owing to its sequence programmability. Herein, a DNA machine based on branched catalytic hairpin assembly ...(bCHA) is introduced for dynamic self-assembly of DNA dendrimers. For this system, a Y-shaped hairpin trimer tethered with three kinds of hairpins (H1, H2 and H3) is constructed. The introduction of an initiator (I) triggers a cascade of CHA reactions among hairpin trimers, leading to the formation of DNA dendrimers. Through labeling fluorophore/quencher pairs in the hairpin trimers, this catalytic DNA machine is applied as a versatile amplification platform to analyze nucleic acids using microRNA-155 (miR-155) as a model analyte. Benefiting from the "diffusion effect", the proposed bCHA achieves a greatly improved sensitivity in comparison with traditional CHA. This catalytic amplifier exhibits high sensitivity toward miR-155 detection with a dynamic range from 2.5 nM to 500 nM and demonstrates excellent selectivity to distinguish the single-base mismatched sequence from the perfectly complementary one, which is further applied to detect low-abundance miR-155 spiked in complex matrices with minimal interference. This method is further applied for
imaging of miR-155 in different live cells. The bCHA reaction can be specifically triggered by intracellular miR-155, achieving monitoring of the dynamic miRNA expression and distribution. Overall, our proposed enzyme-free dynamic DNA self-assembly strategy provides a versatile approach for the development of DNA nanotechnology in biosensing and bioimaging, and monitoring the cellular miRNA-related biological events.
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•DNA-Au nanomachine activated by tumor microenvironment GSH is constructed.•G-quadruplexes are incorporated to target tumor and modulate tumor hypoxia for enhanced PDT.•Triple ...combination of PDT/PTT/chemotherapy is achieved for cancer treatment.
Multimodal therapy is a combination of different therapeutic modalities within one single platform to enhance treatment efficiency, which holds great promise in biomedical research and clinical practice. Herein, a glutathione (GSH)-activated DNA-Au nanomachine with tumor-targeting capacity has been developed for imaging-guided photodynamic/photothermal/chemo combination therapy of murine breast cancer. In this theranostic nanoplatform, G-quadruplex double-stranded DNA (G-dsDNA) containing AS1411 aptamer is served as not only the targeting unit to specifically recognize murine 4T1 breast cancer cells but also the carrier for loading photosensitizer chlorins e6 (Ce6), iron-containing porphyrin hemin and anticancer drug doxorubicin (Dox). In tumor microenvironment, the excessive GSH triggers the rupture of disulfide bridges and results in the dissociation of DNA-Au nanomachine along with the release of Dox for chemotherapy and the aggregation of AuNPs for photothermal therapy (PTT) as well as in situ imaging. Meanwhile, hemin and Ce6 delivered by G-quadruplexes alleviate the tumor hypoxia, which further promotes the generation of reactive oxygen species (ROS) for enhanced photodynamic therapy (PDT). The in vitro and in vivo assays validate that the combination of PDT/PTT/chemotherapy shows a high efficiency for targeted damage of 4T1 cells and inhibition of tumor growth. Therefore, the proposed multifunctional DNA-Au nanomachine provides a powerful and robust theranostic nanoplatform for imaging-guided combinatorial anticancer therapy.
A hyper‐branched hybridization chain reaction (HB‐HCR) is presented herein, which consists of only six species that can metastably coexist until the introduction of an initiator DNA to trigger a ...cascade of hybridization events, leading to the self‐sustained assembly of hyper‐branched and nicked double‐stranded DNA structures. The system can readily achieve ultrasensitive detection of target DNA. Moreover, the HB‐HCR principle is successfully applied to construct three‐input concatenated logic circuits with excellent specificity and extended to design a security‐mimicking keypad lock system. Significantly, the HB‐HCR‐based keypad lock can alarm immediately if the “password” is incorrect. Overall, the proposed HB‐HCR with high amplification efficiency is simple, homogeneous, fast, robust, and low‐cost, and holds great promise in the development of biosensing, in the programmable assembly of DNA architectures, and in molecular logic operations.
DNA branches out: A hyperbranched hybridization chain reaction is reported for the self‐assembly of dendritic DNA structures triggered by an initiator DNA strand. By using this method, ultrasensitive detection of target DNA was achieved and three‐input concatenated logic circuits have been constructed which can operate as keypad locks for biocomputing security systems at the molecular level.
Variation in leaf traits is critical for carbon gains and losses during leaf life and drives litter carbon and nutrient losses via decomposition. Accurately quantifying litter decomposition ...parameters is essential for assessing ecosystem carbon and nutrient dynamics. Leaf litterbags have commonly been employed to measure effects of environmental drivers, decomposers, and plant traits on decomposition rates. There has been much debate regarding the suitability of substituting senesced dead leaves with fresh (green) leaves in litterbags, which has been common practice for mimicking green leaf fall or for practical reasons. Therefore, we tested the null hypothesis that replacement of dead leaves with fresh leaves in litterbag experiments is justified, based on similarities in structural and chemical traits between fresh and dead leaves across plant species and growth forms.
We conducted a paired litterbag decomposition experiment with both fresh and dead leaves of 26 common species in subtropical China, in each of five contrasting ecosystems.
While fresh leaves generally decomposed faster than dead leaves, this deviation varied among species and growth forms, based on their traits. Overall, there was significant but rather weak correlation between dead leaf decomposition rate k and fresh leaf k, across species and ecosystem types; the deviation between fresh and dead leaf k was larger for fast‐decomposing, mostly herbaceous species. The different decomposition patterns for fresh versus dead leaves were underpinned by key underlying traits integrated in leaf resource economics spectra (LES) for fresh and dead leaves. The dead leaf LES exhibited a greater predictive capability for dead leaf k while the fresh leaf LES had higher explanatory value for the fresh leaf k values.
Our findings partly reject the null hypothesis and ask for caution in inferring leaf litter decomposition rates based on green leaf litterbags or traits data. We suggest follow‐up research on substituting senesced roots and stems with fresh ones in decomposition experiments.
Synthesis. Human activities and extreme weather events are leading to increasing pulse inputs of fresh plant parts and our study contributes to knowledge on how they contribute to overall decomposition rates besides senesced litter inputs.
Significant but rather weak correlation exists between leaf decomposition rates (k) of fresh (green) leaves and dead leaves (i.e. litter) across species; the correlation strength varies with growth form (a) but not decomposition site (b). Fresh leaves generally decompose faster than dead ones. Caution is needed in estimating litter decomposition rates using fresh leaf litterbags or fresh leaf traits data.
Plant functional traits are increasingly used to understand ecological relationships and (changing) ecosystem functions. For understanding ecosystem‐level biogeochemistry, we need to understand how ...(much) traits co‐vary between different plant organs across species and its implications for litter decomposition. However, we do not know how the degree of synchronous variation in decomposition rates between organs across species could be influenced by different keystone invertebrates decomposing different senesced plant organs, especially in warm‐climate forests. Here we asked whether interspecific patterns in wood and leaf decomposition rates and in the spectra of resource economics traits underpinning them, co‐vary across woody species; and how (much) the keystone invertebrate decomposers of the litter of these organs enhance or lower such co‐variation of decomposition rates through time.
We addressed these questions through an 18‐month ‘common‐garden’ decomposition experiment using leaf, twig and branch litter of 41 woody species in two distant subtropical forest sites in east China. We quantified the effects of leaf, twig and branch functional traits and their respective key invertebrates (moth larvae, termites) on the decomposition rates of those organs.
Interspecific variation in wood traits was partly decoupled from that in leaf traits across species, while strong coupling was found between twigs and branches. The co‐variation between leaf and woody organ decomposition rates was altered dynamically through the shifting activities of the key decomposers, which created nonlinear relationships of invertebrate litter consumption as a function of species rankings along the resource economic trait spectra of leaves and branches.
The deviations from coupling of decomposition rates between organs were likely caused by combinations of three mechanisms: (1) (de‐)coupling between organs of other traits, not commonly considered in resource economics spectra (e.g. resins) (2) leaf and wood decomposers having specific diet requirements and (3) temporal patterns of the decomposers' activity.
Synthesis. Our study highlights the importance of considering the different ways by which invertebrate detritivores drive decomposition processes through time. Under the ongoing biodiversity decline, future research would benefit from a better understanding of the role of the dynamic interactions between detritivore activities and plant functional traits on the carbon turnover in ecosystems.
Our findings showed that the patterns of coordination between the decomposition rates of leaf and woody organs had been in constant change due to temporal shifts in activity of the key decomposers, highlighting the need to consider the various ways that invertebrate detritivores drive decomposition process over time. Future research would benefit from a better understanding of the interactive effects of dynamic detritivore activity with plant functional traits on the carbon turnovers across ecosystems in the context of ongoing global biodiversity decline.
Toehold-mediated strand displacement-based nanocircuits are developed by integrating catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR), which achieves self-assembly of ...hyperbranched DNA structures and is readily utilized as an enzyme-free amplifier for homogeneous CRET detection of microRNA with high sensitivity and selectivity.
The development of versatile nanotheranostic platforms that integrate both diagnostic and therapeutic functions have always been an intractable challenge in precise cancer treatment. Herein, an ...aptamer‐tethered deoxyribonucleic acids‐gold particle (Apt‐DNA‐Au) nanomachine has been developed for in situ imaging and targeted multimodal synergistic therapy of mammary carcinoma. Upon specifically internalized into MCF‐7 cells, the tumor‐related TK1 mRNA activates the Apt‐DNA‐Au nanomachine by DNA strand displacement cascades, resulting in the release of the fluorophore and antisense DNA as well as the aggregation of AuNPs for in situ imaging, suppression of survivin expression and photothermal therapy, respectively. Meanwhile, the controlled released drugs are used for chemotherapy, while under the laser irradiation the loaded photosensitizer produces reactive oxygen species (ROS) for photodynamic therapy. The results confirm that the proposed Apt‐DNA‐Au nanomachine provides a powerful nanotheranostic platform for in situ imaging‐guided combinatorial anticancer therapy.
A multifunctional DNA‐Au nanomachine which can be triggered by endogenous tumor growth‐related TK1 mRNA has been devised as the combinatorial theranostic agent for fluorescence imaging‐guided chemo, genic, photodynamic, and photothermal synergistic targeted therapy of breast cancer. This theranostic nanoplatform achieves the significant inhibition of tumor growth and improvement of therapeutic efficacy through in situ imaging.
Here we report a target-catalyzed DNA four-way junction (DNA-4WJ) on the basis of toehold-mediated DNA strand displacement reaction (TM-SDR), which is readily applied in enzyme-free amplified ...chemiluminescence resonance energy transfer (CRET) imaging of microRNA. In this system, the introduction of target microRNA-let-7a (miR-let-7a) activates a cascade of assembly steps with four DNA hairpins, followed by a disassembly step in which the target microRNA is displaced and released from DNA-4WJ to catalyze the self-assembly of additional branched junctions. As a result, G-quadruplex subunit sequences and fluorophore fluorescein amidite (FAM) are encoded in DNA-4WJ in a close proximity, stimulating a CRET process in the presence of hemin/K+ to form horseradish peroxidase (HRP)-mimicking DNAzyme that catalyzes the generation of luminol/H2O2 chemiluminescence (CL), which further transfers to FAM. The background signal is easily reduced using magnetic graphene oxide (MGO) to remove unreacted species through magnetic separation, which makes a great contribution to improve the detection sensitivity and achieves a detection limit as low as 6.9 fM microRNA-let-7a (miR-let-7a). In addition, four-input concatenated logic circuits with an automatic reset function have been successfully constructed relying on the architecture of the proposed DNA-4WJ. More importantly, DNA nanohydrogels are self-assembled using DNA-4WJs as building units after centrifugation, which are driven by liquid crystallization and dense packaging of building units. Moreover, the DNA nanohydrogels are readily functionalized by incorporating with aptamers, bioimaging agents, and drug loading sites, which thus are served as efficient nanocarriers for targeted drug delivery and cancer therapy with high loading capacity and excellent biocompatibility.
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