Photocleavable Ortho-Nitrobenzyl-Protected DNA Architectures and Their Applications.

This review article introduces mechanistic aspects and applications of photochemically deprotected ortho-nitrobenzyl (ONB)-functionalized nucleic acids and their impact on diverse research fields including DNA nanotechnology and materials chemistry, biological chemistry, and systems chemistry. Specific topics addressed include the synthesis of the ONB-modified nucleic acids, the mechanisms involved in the photochemical deprotection of the ONB units, and the photophysical and chemical means to tune the irradiation wavelength required for the photodeprotection process. Principles to activate ONB-caged nanostructures, ONB-protected DNAzymes and aptamer frameworks are introduced. Specifically, the use of ONB-protected nucleic acids for the phototriggered spatiotemporal amplified sensing and imaging of intracellular mRNAs at the single-cell level are addressed, and control over transcription machineries, protein translation and spatiotemporal silencing of gene expression by ONB-deprotected nucleic acids are demonstrated. In addition, photodeprotection of ONB-modified nucleic acids finds important applications in controlling material properties and functions. These are introduced by the phototriggered fusion of ONB nucleic acid functionalized liposomes as models for cell-cell fusion, the light-stimulated fusion of ONB nucleic acid functionalized drug-loaded liposomes with cells for therapeutic applications, and the photolithographic patterning of ONB nucleic acid-modified interfaces. Particularly, the photolithographic control of the stiffness of membrane-like interfaces for the guided patterned growth of cells is realized. Moreover, ONB-functionalized microcapsules act as light-responsive carriers for the controlled release of drugs, and ONB-modified DNA origami frameworks act as mechanical devices or stimuli-responsive containments for the operation of DNA machineries such as the CRISPR-Cas9 system. The future challenges and potential applications of photoprotected DNA structures are discussed.

Optical and Electrochemical Probes for Monitoring Cytochrome†c in Subcellular Compartments During Apoptosis.

Cytochrome c (Cyt. c) is a key initiator of the caspases that activate cell apoptosis. The spatiotemporal evaluation of the contents of Cyt. c in cellular compartments and the detection of Cyt. c delivery between cellular compartments upon apoptosis is important for probing cell viabilities. We introduce an optical probe and an electrochemical probe for the quantitative assessment of Cyt. c in cellular compartments at the single cell level. The optical or electrochemical probes are functionalized with photoresponsive o-nitrobenzylphosphate ester-caged Cyt. c aptamer constituents. These are uncaged by light stimuli at single cell compartments, allowing the spatiotemporal detection of Cyt. c through the formation of Cyt. c/aptamer complexes at non-apoptotic or apoptotic conditions. The probes are applied to distinguish the contents of Cyt. c in cellular compartments of epithelial MCF-10A breast cells and malignant MCF-7 and MDA-MB-231 breast cells under apoptotic/non-apoptotic conditions.

Photoactivated Biosensing Process for Dictated ATP Detection in Single Living Cells.

The subcellular distribution of adenosine 5'-triphosphate (ATP) and the concentration of ATP in living cells dynamically fluctuate with time during different cell cycles. The dictated activation of the biosensing process in living cells enables the spatiotemporal target detection in single living cells. Herein, a kind of o-nitrobenzylphosphate ester hairpin nucleic acid was introduced as a photoresponsive DNA probe for light-activated ATP detection in single living cells. Two methods to spatiotemporally activate the probe in single living cells were discussed. One method was the usage of the micrometer-sized optical fiber (about 5 µm) to guide the UV light (λ = 365 nm) to selectively activate the photoresponsive DNA probe in single living cells. The second method involved a two-photon laser confocal scanning microscope to selectively irradiate the photoresponsive DNA probes confined in single living cells via two-photon irradiation (λ = 740 nm). ATP aptamer integrated in the activated DNA probes selectively interacted with the target ATP, resulting in dictated signal generation. Furthermore, the photoactivated biosensing process enables dictated dual-model ATP detection in single living cells with "Signal-ON" fluorescence signal and "Signal-OFF" electrochemical signal outputs. The developed photoactivated biosensor for dictated ATP detection with high spatiotemporal resolution in single living cells at a desired time and desired place suggests the possibility to monitor biomarkers during different cell cycles.

Photoactivated DNA Walker Based on DNA Nanoflares for Signal-Amplified MicroRNA Imaging in Single Living Cells.

Specific and sensitive detection and imaging of cancer-related miRNA in living cells are desirable for cancer diagnosis and treatment. Because of the spatiotemporal variability of miRNA expression level during different cell cycles, signal amplification strategies that can be activated by external stimuli are required to image miRNAs on demand at desired times and selected locations. Herein, we develop a signal amplification strategy termed as the photoactivated DNA walker based on DNA nanoflares, which enables photocontrollable signal amplification imaging of cancer-related miRNA in single living cells. The developed method is achieved via combining photoactivated nucleic acid displacement reaction with the traditional exonuclease III (EXO III)-assisted DNA walker based on DNA nanoflares. This method is capable of on-demand activation of the DNA walker for dictated signal amplification imaging of cancer-related miRNA in single living cells. The developed method was demonstrated as a proof of concept to achieve photoactivated signal amplification imaging of miRNA-21 in single living HeLa cells via selective two-photon irradiation (λ = 740 nm) of single living HeLa cells by using confocal microscopy equipped with a femtosecond laser.

Near-Infrared Light Activated Nucleic Acid Cascade Recycling Amplification for Spatiotemporally Controllable Signal Amplified mRNA Imaging.

The expression level and subcellular distribution of mRNA dynamically changed during the different cell circles. Spatiotemporally controllable signal amplification methods capable of controlling the when and where of the amplification process could allow the sensitive mRNA imaging of selected living cells at dictated time-intervals of the cell life-cycle. However, the present methods for amplified mRNA imaging are hard to control the where and when of the signal amplification due to the lack of an effective strategy to precisely trigger and control the signal amplification process. Herein, we present a conceptual study termed as photocontrollable nucleic acid cascade recycling amplification which uses near-infrared (NIR) light to precisely control and trigger the whole process. This strategy is achieved by integrating photocontrollable nucleic acid displacement reaction with exonuclease III (EXO III) assisted nucleic acid cascade recycling amplification and combination with upconversion nanoparticles (UCNPs), thus resulting in a NIR light activatable signal amplification. As a proof of concept, we demonstrate this developed NIR light triggered signal amplification process in selected living cancer cells for spatiotemporally controllable signal amplified mRNA imaging.

Photoresponsive Electrochemical DNA Biosensors Achieving Various Dynamic Ranges by Using Only-One Capture Probe.

The rational design of DNA capture probes for modulating the binding affinity to tune the dynamic range of electrochemical DNA (E-DNA) biosensors is valuable and effective. Most of current strategies, however, require designing several DNA capture probes to achieve the tunable dynamic range, which is cumbersome and costly. Herein, we develop the photoresponsive E-DNA biosensors with tunable dynamic ranges by using only one photocleavable capture probe (PC-CP). The photoresponsive PC-CP is a stem-loop DNA structure containing a photocleavable linker (PC-linker) in the loop. The PC-linker can be cleaved by UV irradiation to switch the structure of PC-CP, through which the binding affinity to the target could be tuned. In this way, the dynamic range, the sensitivity, and the specificity of photoresponsive E-DNA biosensors can be tuned. Furthermore, the developed photoresponsive E-DNA biosensors enable sensitive and selective detection of target DNA in complex samples with a tunable dynamic range, which offers the possibility of clinical applications.

Two-Photon Lithographic Patterning of DNA-Coated Single-Microparticle Surfaces.

The spatially defined functionalization of microparticles with asymmetric shape-controlled nucleic acid patterns is a major challenge in materials science. The asymmetric patterning of microparticles is important to allow the controlled fabrication of crystalline lattices or controlled aggregates of microparticles. We present the combination of two-photon lithography and photocleavable o-nitrobenzylphosphate ester nucleic acid coating-modified microparticles as a versatile means to asymmetrically pattern single microparticle surfaces. The two-photon patterning of microparticles with predesigned nucleic acid structures of different sizes (700 nm to 2.8 µm) and shapes (circles, rings, triangles, and squares) are demonstrated. In addition, complex patterned domains consisting of two different asymmetric nucleic acid domains are fabricated by the controlled Z-positioning of the microparticles in respect to the two-photon irradiation sources. In addition, the two-photon lithographic patterning of the photocleavable DNA coating allows the generation of functional nucleic acid domains for the photostimulated activation of the catalytic hybridization assembly (CHA) of branched nucleic acid structures on single microparticles.

Triplex-Functionalized DNA Tetrahedral Nanoprobe for Imaging of Intracellular pH and Tumor-Related Messenger RNA.

A new triplex-functionalized DNA tetrahedral nanoprobe is proposed herein for monitoring pH and messenger RNA (mRNA) in living cells. Different from traditional DNA tetrahedron-based nanoprobes, DNA triplex was employed to serve as important conformational conversion elements. Inspired by the low extracellular pH in tumor cells, the mRNA-targeted H1 and H2 were stably assembled on the extended short hairpin probes of DNA tetrahedron via Hoogsteen bonding to form DNA triplex. Due to the high intracellular pH and presence of target mRNA, hybridization chain reaction (HCR) was triggered between H1 and H2 which were released from the dissociation of DNA triplex, and the generated long double-stranded DNA activated a Föster resonance energy transfer (FRET) signal indicating target mRNA expression even at very low contents. By combining the distinguishing feature of DNA triplex structure (pH-responsive) and HCR (signal amplification), sensitive imaging of intracellular pH and tumor-related mRNA can be realized. As a further application, dynamic imaging of intracellular pH and mRNA during "mitochondria-dependent" pathway apoptosis was successfully achieved in human breast cancer cells, which indicated huge potential of our proposed nanoprobe in early diagnosis and treatment of diseases.

Photoactivated Nanoflares for mRNA Detection in Single Living Cells.

Gold nanoparticles (AuNPs) have shown great promise as a universal platform for biosensing and are often functionalized with a densely packed DNA for intracellular detection. While DNA-AuNP conjugates, such as nanoflares, have been used for single and multiple mRNA molecules detection in living cells, the target recognition reaction is triggered once they enter into cells, making it impossible to control the initial reaction at the desired time. To solve this problem, we have designed photoactivated (PA) nanoflares for intracellular mRNA analysis with high spatiotemporal control. PA nanoflares consist of AuNP and photoresponsive DNA hairpin probes. Without UV irradiation, the DNA hairpin could be kept unawakened and show no reactivity to target the probe. Upon UV activation, the hairpin structures are destroyed and expose the sticky domains, which act as toeholds to mediate strand displacement reactions, making flares release from the gold surface and causing an increase of fluorescence. By tuning light irradiation, PA nanoflares for mRNA detection in living cells can be temporally controlled. With the benefit from two-photon laser illumination, PA nanoflares can detect mRNA in selective cells at a desired time point at the single-cell level. Compared to the traditional nanoflares, the novel PA nanoflares have increased the detection sensitivity and achieved intracellular biomarkers detection at the single-cell level with high spatiotemporal control.

Tunable superamphiphobic surfaces: a platform for naked-eye ATP detection.

A superamphiphobic surface composed of two different size ranges of TiO2 nanoparticles was simply fabricated through spraying the perfluorosilane coated TiO2 nanoparticles suspension dispersing in ethanol. The surface chemistry was finely regulated through gradient UV irradiation-induced organic compound degradation to fabricate surface with gradient solid surface energy or wettability. The fabricated surface shows good droplet sorting ability, which can successfully discriminate ethanol droplets with different concentrations. As a proof-of-concept, the biosensor application of this surface was demonstrated by using it for naked-eye ATP detection. Liquid droplets with different concentrations of ATP after ATP-dependent rolling circle amplification (RCA) can be effectively sorted by the surface. This developed biosensor methodology based on droplet sorting ability of the fabricated surface is energy-efficient and economical which is promising for biosensors, point-of-care testing, and biochemical assays. Graphical abstract ᅟ.

Photoactivated Specific mRNA Detection in Single Living Cells by Coupling "Signal-on" Fluorescence and "Signal-off" Electrochemical Signals.

The spatiotemporal detection of a target mRNA in a single living cell is a major challenge in nanoscience and nanomedicine. We introduce a versatile method to detect mRNA at a single living cell level that uses photocleavable hairpin probes as functional units for the optical (fluorescent) and electrochemical (voltammetric) detection of MnSOD mRNA in single MCF-7 cancer cells. The fluorescent probe is composed of an ortho-nitrophenylphosphate ester functionalized hairpin that includes the FAM fluorophore in a caged configuration quenched by Dabcyl. The fluorescent probe is further modified with the AS1411 aptamer to facilitate the targeting and internalization of the probe into the MCF-7 cells. Under UV irradiation, the hairpin is cleaved, leading to the intracellular mRNA toehold-stimulated displacement of the FAM-functionalized strand resulting in a switched-on fluorescence signal upon the detection of the mRNA in a single cell. In addition, a nanoelectrode functionalized with a methylene blue (MB) redox-active photocleavable hairpin is inserted into the cytoplasm of a single MCF-7 cell. Photocleavage of the hairpin leads to the mRNA-mediated toehold displacement of the redox-active strand associated with the probe, leading to the depletion of the voltammetric response of the probe. The parallel optical and electrochemical detection of the mRNA at a single cell level is demonstrated.

Light-Responsive and pH-Responsive DNA Microcapsules for Controlled Release of Loads.

A method to assemble light-responsive or pH-responsive microcapsules loaded with different loads (tetramethylrhodamine-modified dextran, TMR-D; microperoxidase-11, MP-11; CdSe/ZnS quantum dots; or doxorubicin-modified dextran, DOX-D) is described. The method is based on the layer-by-layer deposition of sequence-specific nucleic acids on poly(allylamine hydrochloride)-functionalized CaCO3 core microparticles, loaded with the different loads, that after the dissolution of the core particles with EDTA yields the stimuli-responsive microcapsules that include the respective loads. The light-responsive microcapsules are composed of photocleavable o-nitrobenzyl-phosphate-modified DNA shells, and the pH-responsive microcapsules are made of a cytosine-rich layer cross-linked by nucleic acid bridges. Irradiating the o-nitrobenzyl phosphate-functionalized microcapsules, λ = 365 nm, or subjecting the pH-responsive microcapsules to pH = 5.0, results in the cleavage of the microcapsule shells and the release of the loads. Preliminary studies address the cytotoxicity of the DOX-D-loaded microcapsules toward MDA-MB-231 breast cancer cells and normal MCF-10A breast epithelial cells. Selective cytotoxicity of the DOX-D-loaded microcapsules toward cancer cells is demonstrated.

Reversible Modulation of DNA-Based Hydrogel Shapes by Internal Stress Interactions.

We present the assembly of asymmetric two-layer hybrid DNA-based hydrogels revealing stimuli-triggered reversibly modulated shape transitions. Asymmetric, linear hydrogels that include layer-selective switchable stimuli-responsive elements that control the hydrogel stiffness are designed. Trigger-induced stress in one of the layers results in the bending of the linear hybrid structure, thereby minimizing the elastic free energy of the systems. The removal of the stress by a counter-trigger restores the original linear bilayer hydrogel. The stiffness of the DNA hydrogel layers is controlled by thermal, pH (i-motif), K+ ion/crown ether (G-quadruplexes), chemical (pH-doped polyaniline), or biocatalytic (glucose oxidase/urease) triggers. A theoretical model relating the experimental bending radius of curvatures of the hydrogels with the Young's moduli and geometrical parameters of the hydrogels is provided. Promising applications of shape-regulated stimuli-responsive asymmetric hydrogels include their use as valves, actuators, sensors, and drug delivery devices.

Facile, Fast-Responsive, and Photostable Imaging of Telomerase Activity in Living Cells with a Fluorescence Turn-On Manner.

In situ detecting and monitoring intracellular telomerase activity is significant for cancer diagnosis. In this work, we report a facile and fast-responsive bioprobe for in situ detection and imaging of intracellular telomerase activity with superior photostability. After transfected into living cells, quencher group labeled TS primer (QP) can be extended in the presence of intracellular telomerase. Positive charged TPE-Py molecules (AIE dye) will bind to the primer as well as extension repeated units, producing a telomerase activity-related turn-on fluorescence signal. By incorporating positive charged AIE dye and substrate oligonucleotides, in situ light-up imaging and detection of intracellular telomerase activity were achieved. This strategy exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this facile and fast-responsive telomerase detection method was demonstrated by using it to distinguish tumor cells from normal cells and to monitor the change of telomerase activity during treatment with antitumor drugs, which shows its potential in clinical diagnostic and therapeutic monitoring.

Protease-Responsive Prodrug with Aggregation-Induced Emission Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells.

Controlled drug delivery and real-time tracking of drug release in cancer cells are essential for cancer therapy. Herein, we report a protease-responsive prodrug (DOX-FCPPs-PyTPE, DFP) with aggregation-induced emission (AIE) characteristics for controlled drug delivery and precise tracking of drug release in living cells. DFP consists of three components: AIE-active tetraphenylethene (TPE) derivative PyTPE, functionalized cell penetrating peptides (FCPPs) containing a cell penetrating peptide (CPP) and a short protease-responsive peptide (LGLAG) that can be selectively cleaved by a cancer-related enzyme matrix metalloproteinase-2 (MMP-2), and a therapeutic unit (doxorubicin, DOX). Without MMP-2, this prodrug cannot go inside the cells easily. In the presence of MMP-2, DFP can be cleaved into two parts. One is cell penetrating peptides (CPPs) linked DOX, which can easily interact with cell membrane and then go inside the cell with the help of CPPs. Another is the PyTPE modified peptide which will self-aggregate because of the hydrophobic interaction and turn on the yellow fluorescence of PyTPE. The appearance of the yellow fluorescence indicates the release of the therapeutic unit to the cells. The selective delivery of the drug to the MMP-2 positive cells was also confirmed by using the intrinsic red fluorescence of DOX. Our result suggests a new and promising method for controlled drug delivery and real-time tracking of drug release in MMP-2 overexpression cells.

Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

DNA Polymer Brush Patterning through Photocontrollable Surface-Initiated DNA Hybridization Chain Reaction.

The fabrication of DNA polymer brushes with spatial resolution onto a solid surface is a crucial step for biochip research and related applications, cell-free gene expression study, and even artificial cell fabrication. Here, for the first time, a DNA polymer brush patterning method is reported based on the photoactivation of an ortho-nitrobenzyl linker-embedded DNA hairpin structure and a subsequent surface-initiated DNA hybridization chain reaction (HCR). Inert DNA hairpins are exposed to ultraviolet light irradiation to generate DNA duplexes with two active sticky ends (toeholds) in a programmable manner. These activated DNA duplexes can initiate DNA HCR to generate multifunctional patterned DNA polymer brushes with complex geometrical shapes. Different multifunctional DNA polymer brush patterns can be fabricated on certain areas of the same solid surface using this method. Moreover, the patterned DNA brush surface can be used to capture target molecules in a desired manner.

What Controls the "Off/On Switch" in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

In DNA dynamic nanotechnology, a toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements a displacing action is conjugated on gold nanoparticles. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, i.e., the abrupt jump in behavior of an "off/on switch", in the reaction rate when the invading toehold was extended to eight bases from seven bases. These phenomena are attributed to the effect of steric hindrance arising from the high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.

The combination of gold nanorods and nanoparticles with DNA nanodevices for logic gates construction.

In this work, two DNA nanodevices were constructed utilizing a DNA strand displacement reaction. With the assistance of gold nanoparticles (AuNPs) and gold nanorods (AuNRs), the autonomous reactions can be reflected from the aggregation states of nanoparticles. By sequence design and the two non-overlapping double hump-like UV-vis spectral peaks of AuNPs and AuNRs, two logic gates with multiple inputs and outputs were successfully run with expected outcomes. This method not only shows how to achieve computing with multiple logic calculations but also has great potential for multiple targets detection.

DNA branch migration reactions through photocontrollable toehold formation.

Strand displacement cascades are commonly used to make dynamically assembled structures. Particularly, the concept of "toehold-mediated DNA branch migration reactions" has attracted considerable attention in relation to dynamic DNA nanostructures. However, it is a challenge to obtain and control the formation of pure 1:1 ratio DNA duplexes with toehold structures. Here, for the first time, we report a photocontrolled toehold formation method, which is based on the photocleavage of 2-nitrobenzyl linker-embedded DNA hairpin precursor structures. UV light irradiation (λ ≈ 365 nm) of solutions containing these DNA hairpin structures causes the complete cleavage of the nitrobenzyl linker, and pure 1:1 DNA duplexes with toehold structures are easily formed. Our experimental results indicate that the amount of toehold can be controlled by simply changing the dose of UV irradiation and that the resulting toehold structures can be used for subsequent toehold-mediated DNA branch migration reactions, e.g., DNA hybridization chain reactions. This newly established method will find broad application in the construction of light-powered, controllable, and dynamic DNA nanostructures or large-scale DNA circuits.