Programmed Synthesis by Stimuli‐Responsive DNAzyme‐Modified Mesoporous SiO2 Nanoparticles

DNAzyme‐capped mesoporous SiO₂ nanoparticles (MP SiO₂ NPs) are applied as stimuli‐responsive containers for programmed synthesis. Three types of MP SiO₂ NPs are prepared by loading the NPs with Cy3‐DBCO (DBCO=dibenzocyclooctyl), Cy5‐N₃, and Cy7‐N₃, and capping the NP containers with the Mg²⁺, Zn²⁺, and histidine‐dependent DNAzyme sequences, respectively. In the presence of Mg²⁺ and Zn²⁺ ions as triggers, the respective DNAzyme‐capped NPs are unlocked, leading to the “click” reaction product Cy3‐Cy5. In turn, in the presence of Mg²⁺ ions and histidine as triggers the second set of DNAzyme‐capped NPs is unlocked leading to the Cy3‐Cy7 conjugated product. The unloading of the respective NPs and the time‐dependent formation of the products are followed by fluorescence spectroscopy (FRET). A detailed kinetic model for the formation of the different products is formulated and it correlates nicely with the experimental results.     

DNAzyme- and light-induced dissipative and gated DNA networks

Nucleic acid-based dissipative, out-of-equilibrium systems are introduced as functional assemblies emulating transient dissipative biological transformations. One system involves a Pb²⁺-ion-dependent DNAzyme fuel strand-driven network leading to the transient cleavage of the fuel strand to “waste” products. Applying the Pb²⁺-ion-dependent DNAzyme to two competitive fuel strand-driven systems yields two parallel operating networks. Blocking the competitively operating networks with selective inhibitors leads, however, to gated transient operation of dictated networks, yielding gated catalytic operations. A second system introduces a “non-waste” generating out-of-equilibrium, dissipative network driven by light. The system consists of a trans-azobenzene-functionalized photoactive module that is reconfigured by light to an intermediary state consisting of cis-azobenzene units that are thermally recovered to the original trans-azobenzene-modified module. The cyclic transient photoinduced operation of the device is demonstrated. The kinetic simulation of the systems allows the prediction of the transient behavior of the networks under different auxiliary conditions.

Functional DNA modules are triggered in the presence of appropriate inhibitors to yield transient gated catalytic functions, and a photoresponsive DNA module leads to “waste-free” operation of transient, dissipative dynamic transitions.     

Boronic Acid-Mediated Activity Control of Split 10–23 DNAzymes

The 10–23 DNAzyme is an artificially developed Mg²⁺-dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence-specific fashion. In this study, new split 10–23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5′ end, while the other has a ribonucleotide at its 3′ end, were designed. Herein it is demonstrated that the addition of Mg²⁺ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best-performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.     

Targeted and direct intracellular delivery of native DNAzymes enables highly specific gene silencing

DNAzymes exhibit high potential as gene silencing agents for therapeutic applications. Such purposes, however, are significantly challenged by the targeted and successful delivery of unmodified DNAzymes into cells with minimal side effects. Here, we set out to formulate and demonstrate a new stimuli-responsive and constrained aptamer/DNAzyme (Apt/Dz) catenane nanostructure for highly specific gene silencing. The rational design of the Apt/Dz catenane nanostructure with the respective integration of the aptamer sequence and the completely closed catenane format enables both the targeted capability and significantly improved nuclease resistance, facilitating the stable and targeted delivery of unmodified Dz into cancer cells. Moreover, the Dz enzymatic activity in the constrained structure can only be conditionally regulated by the specific intracellular mRNA sequences to silence the target gene with highly reduced side effects. Results show that the Apt/Dz catenane nanostructure can effectively inhibit the expression of the target gene and the proliferation of cancer cells with high specificity.

The stimuli-responsive and constrained aptamer/DNAzyme catenane nanostructure enables the targeted delivery of native DNAzymes for highly specific gene silencing.     

Metal-ion-dependent folding of a uranyl-specific DNAzyme: insight into function from fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO₂²⁺)‐specific 39E DNAzyme in the presence of Mg²⁺, Zn²⁺, Pb²⁺, or UO₂²⁺. At pH 5.5 and physiological ionic strength (100 mM Na⁺), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg²⁺ or Zn²⁺. However, no folding occurred in the presence of Pb²⁺ or UO₂²⁺; this is analogous to the “lock‐and‐key” catalysis mode first observed in the Pb²⁺‐specific 8–17 DNAzyme. However, Mg²⁺ and Zn²⁺ exert different effects on the 8–17 and 39E DNAzymes. Whereas Mg²⁺ or Zn²⁺‐dependent folding promoted 8–17 DNAzyme activity, the 39E DNAzyme folding induced by Mg²⁺ or Zn²⁺ inhibited UO₂²⁺‐specific activity. Group IIA series of metal ions (Mg²⁺, Ca²⁺, Sr²⁺) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr²⁺ (1.12 Å) is comparable to that of Pb²⁺ (1.20 Å), but contrary to Pb²⁺, Sr²⁺ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb²⁺ and UO₂²⁺. Under low ionic strength (30 mM Na⁺), all four metal ions (Mg²⁺, Zn²⁺, Pb²⁺, and UO₂²⁺), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA‐backbone phosphates in addition to playing specific catalytic roles. Mg²⁺ at low (<2 mM ) concentration promoted UO₂²⁺‐specific activity, whereas Mg²⁺ at high (>2 mM ) concentration inhibited the UO₂²⁺‐specific activity. Therefore, the lock‐and‐key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock‐and‐key mode only at ionic strengths of 100 mM or greater.     

Sharp Switching of DNAzyme Activity through the Formation of a CuII-Mediated Carboxyimidazole Base Pair

DNAzymes are widely used as functional units for creating DNA-based sensors and devices. Switching of DNAzyme activity by external stimuli is of increasing interest. Herein we report a Cu^II-responsive DNAzyme rationally designed by incorporating one of the most stabilizing artificial metallo-base pairs, a Cu^II-mediated carboxyimidazole base pair (Im^C-Cu^II-Im^C), into a known RNA-cleaving DNAzyme. Cleavage of the substrate was suppressed without Cu^II, but the reaction proceeded efficiently in the presence of Cu^II ions. This is due to the induction of a catalytically active structure by Im^C-Cu^II-Im^C pairing. The on/off ratio was as high as 12-fold, which far exceeds that of the previously reported DNAzyme with a Cu^II-mediated hydroxypyridone base pair. The DNAzyme activity can be regulated specifically in response to Cu^II ions during the reaction through the addition, removal, or reduction of Cu^II. This approach should advance the development of stimuli-responsive DNA systems with a well-defined sharp switching function.     

Dynamic Reconfigurable DNA Nanostructures,Networks and Materials

DNA nanotechnology relies on the structural and functional information encoded in nucleic acids. Specifically, the sequence-guided reconfiguration of nucleic acids by auxiliary triggers provides a means to develop DNA switches, machines and stimuli-responsive materials. The present Review addresses recent advances in the construction and applications of dynamic reconfigurable DNA nanostructures, networks and materials. Dynamic transformations proceeding within engineered origami frames or between origami tiles, and the triggered dynamic reconfiguration of scaled supramolecular origami structures are addressed. The use of origami frameworks to assemble dynamic chiroplasmonic optical devices and to operate switchable chemical processes are discussed. Also, the dynamic operation of DNA networks is addressed, and the design of “smart” stimuli-responsive all-DNA materials and their applications are introduced. Future perspectives and applications of dynamic reconfigurable DNA nanostructures are presented.     

The noncovalent dimerization of a G-quadruplex/hemin DNAzyme improves its biocatalytic properties

While many protein enzymes exert their functions through multimerization, which improves both selectivity and activity, this has not yet been demonstrated for other naturally occurring catalysts. Here, we report a multimerization effect applied to catalytic DNAs (or DNAzymes) and demonstrate that the enzymatic efficiency of G-quadruplexes (GQs) in interaction with the hemin cofactor is remarkably enhanced by homodimerization. The resulting non-covalent dimeric GQ–DNAzyme system provides hemin with a structurally defined active site in which both the cofactor (hemin) and the oxidant (H₂O₂) are activated. This new biocatalytic system efficiently performs peroxidase- and peroxygenase-type biotransformations of a broad range of substrates, thus providing new perspectives for biotechnological application of GQs.

Cofactor hemin is sandwiched between 3′ homodimeric G-quadruplexes, leading to an excellent DNAzyme as a mimic of peroxidase and monooxygenase.     

Solvate sponge crystals of (DMF)3NaClO4: reversible pressure/temperature controlled juicing in a melt/press-castable sodium-ion conductor

A new type of crystalline solid, termed “solvate sponge crystal”, is presented, and the chemical basis of its properties are explained for a melt- and press-castable solid sodium ion conductor. X-ray crystallography and atomistic simulations reveal details of atomic interactions and clustering in (DMF)₃NaClO₄ and (DMF)₂NaClO₄ (DMF = N-N′-dimethylformamide). External pressure or heating results in reversible expulsion of liquid DMF from (DMF)₃NaClO₄ to generate (DMF)₂NaClO₄. The process reverses upon the release of pressure or cooling. Simulations reveal the mechanism of crystal “juicing,” as well as melting. In particular, cation–solvent clusters form a chain of octahedrally coordinated Na⁺–DMF networks, which have perchlorate ions present in a separate sublattice space in 3 : 1 stoichiometry. Upon heating and/or pressing, the Na⁺⋯DMF chains break and the replacement of a DMF molecule with a ClO₄⁻ anion per Na⁺ ion leads to the conversion of the 3 : 1 stoichiometry to a 2 : 1 stoichiometry. The simulations reveal the anisotropic nature of pressure induced stoichiometric conversion. The results provide molecular level understanding of a solvate sponge crystal with novel and desirable physical castability properties for device fabrication.

Stimuli-responsive “solvate-sponge”-(DMF)₃NaClO₄ exhibits linear chains of DMF–Na⁺ ions with ClO₄⁻ anions in the interstitial space. At increased pressure or temperature, DMF is expelled (reversibly), resulting in a new stoichiometry-(DMF)₂NaClO₄.     

On the Stability of DNA Origami Nanostructures in Low‐Magnesium Buffers

DNA origami structures have great potential as functional platforms in various biomedical applications. Many applications, however, are incompatible with the high Mg²⁺ concentrations commonly believed to be a prerequisite for maintaining DNA origami integrity. Herein, we investigate DNA origami stability in low‐Mg²⁺ buffers. DNA origami stability is found to crucially depend on the availability of residual Mg²⁺ ions for screening electrostatic repulsion. The presence of EDTA and phosphate ions may thus facilitate DNA origami denaturation by displacing Mg²⁺ ions from the DNA backbone and reducing the strength of the Mg²⁺–DNA interaction, respectively. Most remarkably, these buffer dependencies are affected by DNA origami superstructure. However, by rationally selecting buffer components and considering superstructure‐dependent effects, the structural integrity of a given DNA origami nanostructure can be maintained in conventional buffers even at Mg²⁺ concentrations in the low‐micromolar range.     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.     

An endogenous stimulus detonated nanocluster-bomb for contrast-enhanced cancer imaging and combination therapy

Exploitation of stimuli-responsive nanoplatforms is of great value for precise and efficient cancer theranostics. Herein, an in situ activable “nanocluster-bomb” detonated by endogenous overexpressing legumain is fabricated for contrast-enhanced tumor imaging and controlled gene/drug release. By utilizing the functional peptides as bioligands, TAMRA-encircled gold nanoclusters (AuNCs) endowed with targeting, positively charged and legumain-specific domains are prepared as quenched building blocks due to the AuNCs'' nanosurface energy transfer (NSET) effect on TAMRA. Importantly, the AuNCs can shelter therapeutic cargos of DNAzyme and Dox (Dzs-Dox) to aggregate larger nanoparticles as a “nanocluster-bomb” (AuNCs/Dzs-Dox), which could be selectively internalized into cancer cells by integrin-mediated endocytosis and in turn locally hydrolyzed in the lysosome with the aid of legumain. A “bomb-like” behavior including “spark-like” appearance (fluorescence on) derived from the diminished NSET effect of AuNCs and cargo release (disaggregation) of Dzs-Dox is subsequently monitored. The results showed that the AuNC-based disaggregation manner of the “nanobomb” triggered by legumain significantly improved the imaging contrast due to the activable mechanism and the enhanced cellular uptake of AuNCs. Meanwhile, the in vitro cytotoxicity tests revealed that the detonation strategy based on AuNCs/Dzs-Dox readily achieved efficient gene/chemo combination therapy. Moreover, the super efficacy of combinational therapy was further demonstrated by treating a xenografted MDA-MB-231 tumor model in vivo. We envision that our multipronged design of theranostic “nanocluster-bomb” with endogenous stimuli-responsiveness provides a novel strategy and great promise in the application of high contrast imaging and on-demand drug delivery for precise cancer theranostics.

An in situ activable “nanocluster-bomb” detonated by endogenous overexpressing legumain is fabricated for contrast enhanced cancer imaging and effective gene/chemo-therapy.     

A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing

DNAzymes hold promise for gene‐silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme–MnO₂ nanosystem for gene‐silencing therapy. MnO₂ nanosheets adsorb chlorin e6‐labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit ¹O₂ generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO₂ is reduced to Mn²⁺ ions, which serve as cofactors of 10–23 DNAzyme for gene silencing. The release of Ce6 generates ¹O₂ for more efficient photodynamic therapy. The Mn²⁺ ions also enhance magnetic resonance contrast, providing GSH‐activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.     

Stabilizing DNAzymes through Encapsulation in a Metal–Organic Framework

DNAzymes are a promising class of bioinspired catalyst; however, their structural instability limits their potential. Herein, a method to stabilize DNAzymes by encapsulating them in a metal–organic framework (MOF) host is reported. This biomimetic mineralization process makes DNAzymes active under a wider range of conditions. The concept is demonstrated by encapsulating hemin-G-quadruplex (Hemin-G4) into zeolitic imidazolate framework-90 (ZIF-90), which indeed increases the DNAzyme's structural stability. The stabilized DNAzymes show activities in the presence of Exonuclease I, organic solvents, or high temperature. Owing to its elevated stability and heterogeneous nature, it is possible to perform catalysis under continuous-flow conditions, and the DNAzyme can be reactivated in situ by introducing K⁺. Moreover, it is found that the encapsulated DNAzyme maintains its high enantiomer selectivity, demonstrated by the sulfoxidation of thioanisole to (S)-methyl phenyl sulfoxide. This concept of stabilizing DNAzymes expands their potential application in chemical industry.     

Towards photoswitchable quadruple hydrogen bonds via a reversible “photolocking” strategy for photocontrolled self-assembly

Developing new photoswitchable noncovalent interaction motifs with controllable bonding affinity is crucial for the construction of photoresponsive supramolecular systems and materials. Here we describe a unique “photolocking” strategy for realizing photoswitchable control of quadruple hydrogen-bonding interactions on the basis of modifying the ureidopyrimidinone (UPy) module with an ortho-ester substituted azobenzene unit as the “photo-lock”. Upon light irradiation, the obtained Azo-UPy motif is capable of unlocking/locking the partial H-bonding sites of the UPy unit, leading to photoswitching between homo- and heteroquadruple hydrogen-bonded dimers, which has been further applied for the fabrication of novel tunable hydrogen bonded supramolecular systems. This “photolocking” strategy appears to be broadly applicable in the rational design and construction of other H-bonding motifs with sufficiently photoswitchable noncovalent interactions.

A photolocking strategy is described to achieve the construction of effectively photoswitchable quadruple hydrogen bonds featuring with photoregulable H-bonding affinities, which is further applied in the photocontrollable H-bonded self-assemblies.     

Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells

DNAzymes, which are sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Despite their significant promise, cellular sensing using DNAzymes has however been difficult, mainly because of the “always‐on” mode of first‐generation DNAzyme sensors. To overcome this limitation, a photoactivatable (or photocaged) DNAzyme was designed and synthesized, and its application in sensing Zn^II in living cells was demonstrated. In this design, the adenosine ribonucleotide at the scissile position of the 8–17 DNAzyme was replaced by 2′‐O‐nitrobenzyl adenosine, rendering the DNAzyme inactive and thus allowing its delivery into cells intact, protected from nonspecific degradation within cells. Irradiation at 365 nm restored DNAzyme activity, thus allowing the temporal control over the sensing activity of the DNAzyme for metal ions. The same strategy was also applied to the GR‐5 DNAzyme for the detection of Pb^II, thus demonstrating the possible scope of the method.     

Modulation of DNAzyme Activity via Butanol Dehydration

Understanding the activity of biomolecules in cosolvent systems is important for catalysis, separation and developing biosensors. The majority of previously studied solvents are either phase separated with water or miscible with water. Butanol was recently used to extract water for the conjugation of DNA to gold nanoparticles. In this work, the effect of butanol on the activity of a few RNA-cleaving DNAzymes was studied. A 130-fold improvement in sensitivity for the Na⁺-specific EtNa DNAzyme was observed, and butanol also improved the activity of another Na⁺-specific DNAzyme, NaA43T by a few folds. However, when divalent metal ions were used for both EtNa and 17E DNAzymes, the activity was inhibited. A main driven force for enhanced DNAzyme activity is the concentration effect due to butanol dehydration. This study provides insights into the interplay between DNA, metal ions and organic solvents, and such an understanding might be useful for developing sensitive biosensors.     

A protecting group strategy to access stable lacunary polyoxomolybdates for introducing multinuclear metal clusters

Although metal-containing polyoxomolybdates (molybdenum oxide clusters) exhibit outstanding catalytic properties, their precise synthetic method has not yet been developed. This is mainly because the very low stability of the multivacant lacunary polyoxomolybdates limited their use as synthetic precursors. Here, we present a “protecting group strategy” in polyoxometalate synthesis and successfully develop an efficient method for synthesising multinuclear metal-containing polyoxomolybdates using pyridine as a protecting group for unstable trivacant lacunary Keggin-type polyoxomolybdate [PMo₉O₃₄]⁹⁻. Specifically, tetranuclear cubane- and planar-type manganese clusters were selectively synthesised in the polyoxomolybdates using the present method. The importance of this work is that, in addition to being the first practical way of utilizing multivacant lacunary polyoxomolybdates as precursors, this new “protecting group strategy” will make it possible to produce polyoxometalates with unexplored structures and properties.

A “protecting group strategy” for unstable lacunary polyoxomolybdates enabled successful synthesis of two types of tetranuclear manganese clusters.     

DNAzyme‐Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal‐binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg²⁺‐specific 10–23 or Zn²⁺‐specific 8–17 RNA‐cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg²⁺ or Zn²⁺ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal‐specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein‐based sensors.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).