Construction of an exogenously and endogenously Co-activated DNA logic amplifier for highly reliable intracellular MicroRNA imaging.

DNA-based molecular amplifiers offer significant promise for molecular-level disease diagnosis and treatment, yet tailoring their activation for precise timing and localization remains a challenge. Herein, we've pioneered a dual activation strategy harnessing external light and internal ATP to create a highly controlled DNA logic amplifier (FDLA) for accurate miRNA monitoring in cancer cells. The FDLA was constructed by tethered the two functionalized catalytic hairpin assembly (CHA) hairpin modules (ATP aptamer sealed hairpin aH1 and photocleavable (PC-linker) sites modified hairpin pH2) to DNA tetrahedron (DTN). The FDLA system incorporates ATP aptamers and PC-linkers as logic control units, allowing them to respond to both exogenous UV light and endogenous ATP present within cancer cells. This response triggers the release of CHA hairpin modules, enabling amplified FRET miRNA imaging through an AND-AND gate. The DTN structure could improve the stability of FDLA and accelerate the kinetics of the strand displacement reaction. It is noteworthy that the UV and ATP co-gated DNA circuit can control the DNA bio-computing at specific time and location, offering spatial and temporal capabilities that can be harnessed for miRNA imaging. Furthermore, the miRNA-sensing FDLA amplifier demonstrates reliable imaging of intracellular miRNA with minimal background noise and false-positive signals. This highlights the feasibility of utilizing both exogenous and endogenous regulatory strategies to achieve spatial and temporal control of DNA molecular circuits within living cancer cells. Such advancements hold immense potential for unraveling the correlation between miRNA and associated diseases.

A smart metal-polyphenol-DNAzyme nanoplatform for gene-chemodynamic synergistic tumor therapy.

DNAzyme-based gene regulation shows great potential for the therapy of many cancers. However, ineffective delivery and insufficient cofactor supply pose challenges for potent gene therapy. In this study, we constructed a smart metal-polyphenol-DNAzyme nanoplatform (TA-Mn@Dz NPs) with intrinsic stability, effective delivery, and cofactor self-supply ability for gene-chemodynamic synergistic tumor therapy. Tannic acid, a plant-derived polyphenol, acts as an intermediate structural unit to mediate the assembly of Mn2+/DNAzyme and tumor acid environment-responsive nanocarriers. Intracellularly, the acidic environment triggers the decomposition of TA-Mn@Dz NPs to release DNAzyme and Mn2+. The Mn2+ ion not only boosts the catalytic cleavage of surviving mRNA for effective gene therapy but also activates chemodynamic therapy (CDT), generating highly toxic ·OH from endogenous H2O2. When tail intravenously injected into MCF-7 tumor-bearing mice, the TA-Mn@Dz NPs display desirable synergistic gene-chemodynamic antitumor effects, paving the way for developing DNAzyme-based multifunctional theranostic platforms for biomedical applications. STATEMENT OF SIGNIFICANCE: 1. A smart metal-polyphenol-DNAzyme nanoplatform was constructed for gene-chemodynamic synergistic tumor therapy. 2. Tannic acid act as intermediate structural units to mediate the assembly of Mn2+/DNAzyme and tumor acid environment-responsive nanocarriers. 3. The Mn2+-ion could not only boost the catalytic cleavage of surviving mRNA for effective gene therapy, but also catalyze endogenous H2O2 to form cytotoxic hydroxyl radicals for chemodynamic therapy. 4. Our work paves an extremely simple way to integrate gene therapy with CDT for the dual-catalytic tumor treatment.

In situ continuous Dopa supply by responsive artificial enzyme for the treatment of Parkinson's disease.

Oral dihydroxyphenylalanine (Dopa) administration to replenish neuronal dopamine remains the most effective treatment for Parkinson's disease (PD). However, unlike the continuous and steady dopamine signaling in normal neurons, oral Dopa induces dramatic fluctuations in plasma Dopa levels, leading to Dopa-induced dyskinesia. Herein, we report a functional nucleic acid-based responsive artificial enzyme (FNA-Fe3O4) for in situ continuous Dopa production. FNA-Fe3O4 can cross the blood-brain barrier and target diseased neurons relying on transferrin receptor aptamer. Then, FNA-Fe3O4 responds to overexpressed α-synuclein mRNA in diseased neurons for antisense oligonucleotide treatment and fluorescence imaging, while converting to tyrosine aptamer-based artificial enzyme (Apt-Fe3O4) that mimics tyrosine hydroxylase for in situ continuous Dopa production. In vivo FNA-Fe3O4 treatment results in recovery of Dopa and dopamine levels and decrease of pathological overexpressed α-synuclein in PD mice model, thus ameliorating motor symptoms and memory deficits. The presented functional nucleic acid-based responsive artificial enzyme strategy provides a more neuron friendly approach for the diagnosis and treatment of PD.

Lanthanide-based luminescent probes for biological magnesium: accessing polyphosphate-bound Mg2.

Biomolecule-bound Mg2+ species, particularly polyphosphate complexes, represent a large and dynamic fraction of the total cellular magnesium that is essential for cellular function but remains invisible to most indicators. Here we report a new family of Eu(III)-based indicators, the MagQEu family, functionalized with a 4-oxo-4H-quinolizine-3-carboxylic acid metal recognition group/sensitization antenna for turn-on, luminescence-based detection of biologically relevant Mg2+ species.

Intracellular miRNA Imaging Based on a Self-Powered and Self-Feedback Entropy-Driven Catalyst-DNAzyme Circuit.

DNAzyme-based signal amplification circuits promote the advances in low-abundant miRNA imaging in living cells. However, due to the insufficient cofactor in living cells and unsustainable target utilization, self-powered and self-feedback DNAzyme amplification circuits have rarely been achieved. Here, a MnO2 nanosheet-mediated self-powered and self-feedback entropy-driven catalyst (EDC)-DNAzyme nanoprobe (MnPFEDz) was demonstrated for sensitive imaging of intracellular microRNA (miRNA). In this strategy, MnPFEDz was formed by adsorbing EDC modules and substrate probes on MnO2 nanosheets. The MnO2 nanosheets acted not only as glutathione (GSH)-responsive nanocarriers for efficient delivery of DNA probes but also as a DNAzyme cofactor supplier to power the DNAzyme biocatalysis and promote signal transduction in a feedback way. When entering the cells, GSH could decompose MnO2 nanosheets to generate numerous Mn2+ ion cofactors, leading to the release of DNA probes. Subsequently, the target miRNA initiated EDC cycles to generate amplified fluorescence signals and exposed the complete DNAzyme. Meanwhile, each of the exposed DNAzyme then cleaved the substrate probes with the help of Mn2+ ion cofactors and released a new trigger analogue for the next round of EDC cycles, initiating additional fluorescence signals in a feedback way. As a multiple signal amplification strategy, the MnPFEDz nanoprobe facilitated the effective detection of intracellular molecules with enhanced sensitivity and provided a versatile strategy for the construction of self-powered and self-feedback DNA circuits in living cells.

An aptamer-tethered DNA origami amplifier for sensitive and accurate imaging of intracellular microRNA.

Accurate detection and imaging of low-abundance microRNA (miRNA) in living cells are essential for the diagnosis and prognosis of diseases. Designing nanoprobes with resistance to enzyme degradation, effective cell-binding, and efficient signal amplification is crucial for in vivo imaging. In this study, we present an aptamer-tethered DNA origami amplifier (ADOA) that functions inside living cells to detect miRNA with high sensitivity and stability. In the design, cancer cell-targeting aptamers were tethered onto the border of the DNA origami to improve the discrimination between cancer cells and normal cells. Two substrate modules for the intramolecular entropy-driven reaction (EDR) circuit were alternately arranged on the DNA origami plane. The target miRNA will initiate the sequential hybridization of the two substrate modules on the DNA origami, generating amplified fluorescence signals. The proposed ADOA achieved an accelerated cascade reaction due to the "confinement effect" and significantly enhanced the sensitivity compared with a traditional EDR. Meanwhile, with the rigid structure of the DNA origami, the ADOA possessed excellent signalling stability in living cells. Therefore, the ADOA could expand the application of DNA origami in miRNA sensing and has potential value in early-stage clinical diagnosis.

Target-driven assembly of DNAzyme probes for simultaneous electrochemical detection of multiplex microRNAs.

In this work, we employed target-driven assembly of a Mg2+-dependent DNAzyme to develop an ultrasensitive electrochemical biosensor for the simultaneous detection of miRNA-21 and miRNA-141. The target miRNAs could hybridize with two partial DNAzymes, facilitating the formation of a stable and active Mg2+-dependent DNAzyme. With the help of the Mg2+ cofactor, the DNAzyme could circularly cleave the ferrocene (Fc) or methylene blue (MB) labelled hairpin probes and release Fc and MB labels from the electrode surface, which could significantly amplify the current suppression to achieve multiple detection of small amounts of miRNA-21 and miRNA-141. This electrochemical biosensor showed high sensitivity and selectivity for the simultaneous detection of miRNA-21 and miRNA-141. Furthermore, the proposed method was also successfully applied for the determination of miRNA-21 and miRNA-141 from diluted serum samples. Overall, the proposed sensor showed several considerable advantages including simple preparation, high sensitivity, and enzyme-free signal amplification. Therefore, the proposed electrochemical biosensor could be used as a highly efficient amplification strategy for simultaneous detection of various miRNA biomarkers in bioanalysis and clinical diagnostics.

Highly Selective, Red Emitting BODIPY-Based Fluorescent Indicators for Intracellular Mg2+ Imaging.

Most fluorescent indicators for Mg2+ suffer from poor selectivity against other divalent cations, especially Ca2+, thus do not provide reliable information on cellular Mg2+ concentrations in processes in which such metals are involved. We report a new set of highly selective fluorescent indicators based on alkoxystyryl-functionalized BODIPY fluorophores decorated with a 4-oxo-4H-quinolizine-3-carboxylic acid metal binding moiety. The new sensors, MagQ1 and MagQ2, display absorption and emission maxima above 600 nm, with a 29-fold fluorescence enhancement and good quantum yields (Φ > 0.3) upon coordination of Mg2+ in aqueous buffer. Fluorescence response to Mg2+ is not affected by the presence of competing divalent cations typically present in the cellular milieu, and displays minimal pH dependence in the physiologically relevant range. The choice of alkoxy groups decorating the styryl BODIPY core does not influence the basic photophysical and metal binding properties of the compounds, but has a marked effect on their intracellular retention and thus in their applicability for detection of cellular Mg2+ by fluorescence imaging. In particular, we demonstrate the utility of a triethyleneglycol (TEG) functionalization tactic that endows MagQ2 with superior cellular retention in live cells by reducing active extrusion through organic anion transporters, which are thought to cause fast leakage of typical anionic dyes. With enhanced retention and excellent photophysical properties, MagQ2 can be applied in the detection of cellular Mg2+ influx without interference of high concentrations of Ca2+ akin to those involved in signaling.

Bright, red emitting fluorescent sensor for intracellular imaging of Mg2+ .

Fluorescent sensors with low-energy excitation are in great demand for the study of cellular Mg2+ by fluorescence miscroscopy, but to date they remain scarce. Addressing this gap, we report herein a new set of molecular fluorescent sensors for the detection of Mg2+ based on an o-aminophenol-N,N,O-triacetic acid (APTRA) metal-recognition moiety combined with two different BODIPY fluorophores. The new sensors, MagB1 and MagB2, display absorption and emission maxima in the visible range and respond to Mg2+ in aqueous buffer with large fluorescence enhancements. MagB2, a red-emitting fluorescent indicator based on a styryl-BODIPY, displays superior metal selectivity and optical properties compared to its green emitting counterpart, MagB1. With an excellent 58-fold fluorescence turn-on and Mg2+ dissociation constant in tune with physiological concentrations of the cation (low millimolar range), MagB2 enables visualization of changes in intracellular levels of free Mg2+ in live cells with no significant interference from basal levels of Ca2+, the most common competitor.

A dinuclear ruthenium(II) complex as a one- and two-photon luminescent probe for biological Cu(2+) detection.

A new dinuclear Ru(II) polypyridyl complex, [(bpy)2 Ru(H2 bpip)Ru(bpy)2 ](4+) (RuH2 bpip, bpy=2,2-bipyridine, H2 bpip=2,6-pyridyl(imidazo[4,5-f][1,10]phenanthroline), was developed to act as a one- and two-photon luminescent probe for biological Cu(2+) detection. This Ru(II) complex shows a significant two-photon absorption cross section (400 GM) and displays a remarkable one- and two-photon luminescence switch in the presence of Cu(2+) ions. Importantly, RuH2 bpip can selectively recognise Cu(2+) in aqueous media in the presence of other abundant cellular cations (such as Na(+) , K(+) , Mg(2+) , and Ca(2+) ), trace metal ions in organisms (such as Zn(2+) , Ag(+) , Fe(3+) , Fe(2+) , Ni(2+) , Mn(2+) , and Co(2+) ), prevalent toxic metal ions in the environment (such as Cd(2+) , Hg(2+) , and Cr(3+) ), and amino acids, with high sensitivity (detection limit≤3.33×10(-8) M) and a rapid response time (≤15 s). The biological applications of RuH2 bpip were also evaluated and it was found to exhibit low cytotoxicity, good water solubility, and membrane permeability; RuH2 bpip was, therefore, employed as a sensing probe for the detection of Cu(2+) in living cells and zebrafish.

Synthesis, characterization, and DNA-binding studies of ruthenium complexes [Ru(tpy)(ptn)]2+ and Ru(dmtpy)(ptn)]2+.

Two ruthenium(II) polypyridyl complexes [Ru(tpy)(ptn)](2+) (1) and Ru(dmtpy)(ptn)](2+) (2) (ptn=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]naphthalene, tpy=2,2':6',2"-terpyridine, dmtpy=5,5'-dimethyl-2,2':6',2"-terpyridine) have been synthesized and characterized by elemental analysis, (1)H NMR, mass spectrometry and crystal structure analysis. Spectroscopic studies together with isothermal titration calorimetry (ITC) and viscosity measurements prove that two complexes bind to DNA in an intercalative mode. ITC experiments show that the binding mode for complex 2 is entropically driven, while an entropy-driven initial binding of complex 1 is followed by an entropically and enthalpically favorable process. This difference may be attributed to the ancillary ligand effects on the DNA binding of Ru(II) complexes. Circular dichroism titrations of calf thymus DNA (CT-DNA) with Ru(II) complexes show that complexes 1 and 2 induce B to Z conformational transition of calf thymus DNA at low ionic strength (0.05 M NaCl). The induced Z-DNA conformation can revert to B form when Ru(II) complexes are displaced by ethidium bromide or at high ionic strengths ([NaCl]=0.4 M), but keeps intact with temperature ranged from 25 to 90 °C. The unique structure and characteristics of Ru(II) complexes designed in this investigation will be useful for the study of Z-DNA.