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.

Biomacromolecule-Functionalized AIEgens for Advanced Biomedical Studies.

The advances in bioinformatics and biomedicine have promoted the development of biomedical imaging and theranostic systems to respectively extend the endogenous biomarker imaging with high contrast and enhance the therapeutic effect with high efficiency. The emergence of biomacromolecule-functionalized aggregation-induced emitters (AIEgens), utilizing AIEgens, and biomacromolecules (nucleic acids, peptides, glycans, and lipids), displays specific targeting ability to cancer cell, improved biocompatibility, reduced toxicity, enhanced therapeutic effect, and so forth. This review summarizes the rational design of biomacromolecule-functionalized AIEgens and their biomedical applications in recent ten years, including high-resolution optical imaging of cell, tissue, and small animal model with low background; the biomarker detection for early diagnosis and prognosis; the delivery and monitoring of prodrugs; image-guide photodynamic therapy and its combination with chemotherapy. Through illustrating their functional mechanisms and application, it is hoped that this review would open up a completely new train of research thought for attracted researchers in various fields.

Self-Replicating Catalyzed Hairpin Assembly for Rapid Signal Amplification.

A rapid signal amplification system based on the self-replicating catalyzed hairpin assembly is reported in which two hairpins, H1 and H2, were well-designed in which two split target/trigger DNA and two split G-quadruplex sequences were respectively integrated into H1 and H2. Target/trigger DNA can be cyclically used in this system to form the duplex DNA assemblies (H1-H2), which will bring the two G-quadruplex fragments into close-enough proximity to induce the formation of intact G-quadruplex as a colorimetric signal readout. Similarly, the two split target/trigger DNA sequences will reunite into a DNA sequence that is identical to the target/trigger DNA; then, the obtained replica can also be cyclically used as a new activator unit to trigger the CHA reaction, leading to the rapidly and significantly enhanced formation of target/trigger DNA replicas with the concomitant generation of a higher signal. This self-replication-based autocatalytic signal amplified approach has been successfully used to develop a rapid and visual assay for DNA and small molecule detection.

Sensitive determination of sulfonamides in environmental water by capillary electrophoresis coupled with both silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector.

A new detector, silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector (SDW-ICOF-LED-IFD), is introduced for capillary electrophoresis (CE). The strategy of the work was that half surface of the detection window was coated with silver mirror, which could reflect the undetected fluorescence to the photomultiplier tube to be detected, consequently enhancing the detection sensitivity. Sulfonamides (SAs) are important antibiotics that achieved great applications in many fields. However, they pose a serious threat on the environment and human health when they enter into the environment. The SDW-ICOF-LED-IFD-CE system was used to determine fluorescein isothiocyanate (FITC)-labeled sulfadoxine (SDM), sulfaguanidine (SGD) and sulfamonomethoxine sodium (SMM-Na) in environmental water. The detection results obtained by the SDW-ICOF-LED-IFD-CE system were compared to those acquired by the CE with in-capillary optical fiber light-emitting diode-induced fluorescence detection (ICOF-LED-IFD-CE). The limits of detection (LODs) of SDW-ICOF-LED-IFD-CE and ICOF-LED-IFD-CE were 1.0-2.0 nM and 2.5-7.7 nM (S/N = 3), respectively. The intraday (n = 6) and interday (n = 6) precision of migration time and corresponding peak area for both types of CE were all less than 0.86% and 3.68%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 92.5-102.9%. The results indicated that the sensitivity of the SDW-ICOF-LED-IFD-CE system was improved, and that its reproducibility and accuracy were satisfactory. It was successfully applied to analyze SAs in environmental water.

Rapid DNA detection based on self-replicating catalyzed hairpin assembly using nucleotide base analog pyrrolo-deoxycytidine as fluorophore.

A rapid signal amplified DNA detection method based on self-replicating catalyzed hairpin assembly (SRCHA) has been proposed. In this SRCHA system, two split target DNA sequences were respectively integrated into hairpin auxiliary probes H1 and H2. H2 was used as fluorescent probe which containing a fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) at the end of the stem. Target DNA can be circularly used in this SRCHA system to form the helix DNA H1-H2 complex, the structure change of H2 will move P-dC from hairpin stem to flexible ssDNA sticky end, leading to fluorescence increase due to the less stacking interaction. Meanwhile, the two spilt target DNA sequence was reunited and the target DNA replicate was obtained, which also can be circularly used as new activator to trigger additional CHA reaction and fluorescence signal was then rapidly and significantly enhanced. This SRCHA system has been successfully employed for DNA detection with picomolar within around 15min, and provides a potential technology for the real-time rapid bioanalysis.

Self-assembly of DNA nanoparticles through multiple catalyzed hairpin assembly for enzyme-free nucleic acid amplified detection.

It is known that DNA molecules can be used to build a various of complicated geometrical DNA nanostructures with programmable sequence design, and these DNA nanomaterials show a promising application in biotechnology and biomedicine. However, the construction of large-sized three dimensional DNA-based nanomaterials still remains a challenge. In this work, we propose a new strategy that only employs one target DNA to trigger multiple catalyzed hairpin assembly (CHA) reactions and sticky ends self-assembly to prepare hundreds of nanometer-sized DNA nanoparticles. Moreover, the obtained DNA nanoparticles can be served as efficient biosensors for sensitive colorimetric nucleic acids detection with a detection limit of 7.7pM.

Integrated multi-ISE arrays with improved sensitivity, accuracy and precision.

Increasing use of ion-selective electrodes (ISEs) in the biological and environmental fields has generated demand for high-sensitivity ISEs. However, improving the sensitivities of ISEs remains a challenge because of the limit of the Nernstian slope (59.2/n mV). Here, we present a universal ion detection method using an electronic integrated multi-electrode system (EIMES) that bypasses the Nernstian slope limit of 59.2/n mV, thereby enabling substantial enhancement of the sensitivity of ISEs. The results reveal that the response slope is greatly increased from 57.2 to 1711.3 mV, 57.3 to 564.7 mV and 57.7 to 576.2 mV by electronic integrated 30 Cl- electrodes, 10 F- electrodes and 10 glass pH electrodes, respectively. Thus, a tiny change in the ion concentration can be monitored, and correspondingly, the accuracy and precision are substantially improved. The EIMES is suited for all types of potentiometric sensors and may pave the way for monitoring of various ions with high accuracy and precision because of its high sensitivity.

Nucleotide base analog pyrrolo-deoxycytidine as fluorescent probe signal for enzyme-free and signal amplified nucleic acids detection.

In the present work, an enzyme-free and signal amplified nucleic acids detection method based on the fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) and catalyzed hairpin assembly (CHA) has been developed. In the CHA signal amplification system, two hairpin auxiliary probes, H1 and H2, which containing a fluorescent P-dC at the end of the stem, respectively, were used as the fluorescent probes. The fluorescence of P-dC in H1 and H2 was quenched owing to the stacking interaction among the bases in the stem. In the presence of the target DNA, the catalytic assembly of H1 and H2 was triggered and the target could be released during the helix DNA H1-H2 complex formation process, then the released target was used to trigger another reaction cycle. In the H1-H2 complex, P-dC was located in the flexible single-stranded DNA (ssDNA) sticky ends instead of the rigid stem, thus resulting in the increase of fluorescence. The cycling use of the target in the CHA system amplified the fluorescence signal, and the detection limit of this method was obtained as 19pM, which is 3 orders of magnitude sensitive than the conventional fluorescent nucleotide base analogs based approach without CHA signal amplification.

Unusual sequence length-dependent gold nanoparticles aggregation of the ssDNA sticky end and its application for enzyme-free and signal amplified colorimetric DNA detection.

It is known that the adsorption of short single-stranded DNA (ssDNA) on unmodified gold nanoparticles (AuNPs) is much faster than that for long ssDNA, and thus leads to length-dependent AuNPs aggregation after addition of salt, the color of the solutions sequentially changed from red to blue in accordance with the increase of ssDNA length. However, we found herein that the ssDNA sticky end of hairpin DNA exhibited a completely different adsorption behavior compared to ssDNA, an inverse blue-to-red color variation was observed in the colloid solution with the increase of sticky end length when the length is within a certain range. This unusual sequence length-dependent AuNPs aggregation might be ascribed to the effect of the stem of hairpin DNA. On the basis of this unique phenomenon and catalytic hairpin assembly (CHA) based signal amplification, a novel AuNPs-based colorimetric DNA assay with picomolar sensitivity and specificity was developed. This unusual sequence length-dependent AuNPs aggregation of the ssDNA sticky end introduces a new direction for the AuNPs-based colorimetric assays.

Target-catalyzed autonomous assembly of dendrimer-like DNA nanostructures for enzyme-free and signal amplified colorimetric nucleic acids detection.

Self-assembly of DNA nanostructures is of great importance in nanomedicine, nanotechnology and biosensing. Herein, a novel target-catalyzed autonomous assembly pathway for the formation of dendrimer-like DNA nanostructures that only employing target DNA and three hairpin DNA probes was proposed. We use the sticky-ended Y shape DNA (Y-DNA) as the assembly monomer and it was synthesized by the catalyzed hairpin assembly (CHA) instead of the DNA strand annealing method. The formed Y-DNA was equipped with three ssDNA sticky ends and two of them were predesigned to be complementary to the third one, then the dendrimer-like DNA nanostructures can be obtained via an autonomous assembly among these sticky-ended Y-DNAs. The resulting nanostructure has been successfully applied to develop an enzyme-free and signal amplified gold nanoparticle (AuNP)-based colorimetric nucleic acids assay.

Target-triggered autonomous assembly of DNA polymer chains and its application in colorimetric nucleic acid detection.

A novel target-triggered DNA autonomous assembly pathway for the formation of one-dimensional DNA polymer chains was proposed based on the catalyzed hairpin assembly and sticky end self-assembly, which led to an enzyme-free and signal amplified gold nanoparticle (AuNP) based colorimetric nucleic acid assay with picomolar sensitivity and specificity.