Gene Expression on DNA Biochips Patterned with Strand‐Displacement Lithography

Lithographic patterning of DNA molecules enables spatial organization of cell‐free genetic circuits under well‐controlled experimental conditions. Here, we present a biocompatible, DNA‐based resist termed “Bephore”, which is based on commercially available components and can be patterned by both photo‐ and electron‐beam lithography. The patterning mechanism is based on cleavage of a chemically modified DNA hairpin by ultraviolet light or electrons, and a subsequent strand‐displacement reaction. All steps are performed in aqueous solution and do not require chemical development of the resist, which makes the lithographic process robust and biocompatible. Bephore is well suited for multistep lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision. As an application, we demonstrate compartmentalized, on‐chip gene expression from three sequentially immobilized DNA templates, leading to three spatially resolved protein‐expression gradients.     

Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.     

Light‐Controlled Formation of Vesicles and Supramolecular Organogels by a Cholesterol‐Bearing Amphiphilic Molecular Switch

A new responsive material composed of an amphiphilic light‐switchable dithienylethene unit functionalized with a hydrophobic cholesterol unit and a hydrophilic poly(ethylene glycol)‐modified pyridinium group has been designed. This unique single‐molecule system shows responsive light‐switchable self‐assembly in both water and organic solvents. Light‐triggered reversible vesicle formation in aqueous solutions is reported. The molecule shows a different behavior in apolar aromatic solvents, in which light‐controlled formation of organogel fibers is observed. The light‐triggered aggregation behavior of this molecule demonstrates that control of a supramolecular structure with light can be achieved in both aqueous and organic media and that this ability can be present in a single molecule. This opens the way toward the effective development of new strategies in soft nanotechnology for applications in controlled chemical release systems.     

A DNA‐Mediated Chemically Induced Dimerization (D‐CID) Nanodevice for Nongenetic Receptor Engineering To Control Cell Behavior

Small‐molecule regulation is a powerful switching tool to manipulate cell signal transduction for a desired function; however, most available methods usually require genetic engineering to endow cells with responsiveness to user‐defined small molecules. Herein, we demonstrate a nongenetic approach for small‐molecule‐controlled receptor activation and consequent cell behavior manipulation that is based on DNA‐mediated chemically induced dimerization (D‐CID). D‐CID uses a programmable chemical‐responsive DNA nanodevice to trigger DNA strand displacement and induce the activation of c‐Met, a tyrosine kinase receptor cognate for hepatocyte growth factor, through dimerization. Through the use of various functional nucleic acids, including aptamers and DNAzymes, as recognition modules, the versatility of D‐CID in inducing c‐Met signaling upon addition of various small‐molecular or ionic cues, including ATP, histidine, and Zn²⁺, is demonstrated. Moreover, owing its multi‐input properties, D‐CID can be used to manipulate the behaviors of multiple cell populations simultaneously in a selective and programmable fashion.     

Yoctoliter Thermometry for Single‐Molecule Investigations: A Generic Bead‐on‐a‐Tip Temperature‐Control Module

A new temperature‐jump (T‐jump) strategy avoids photo‐damage of individual molecules by focusing a low‐intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T‐jump of 65 °C within milliseconds. To measure the temperature in situ in single‐molecule experiments, the temperature‐dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead‐on‐a‐tip module and the robust single‐molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T‐jump and single‐molecule techniques.     

Logic‐Gate‐Actuated DNA‐Controlled Receptor Assembly for the Programmable Modulation of Cellular Signal Transduction

Programming cells to sense multiple inputs and activate cellular signal transduction cascades is of great interest. Although this goal has been achieved through the engineering of genetic circuits using synthetic biology tools, a nongenetic and generic approach remains highly demanded. Herein, we present an aptamer‐controlled logic receptor assembly for modulating cellular signal transduction. Aptamers were engineered as “robotic arms” to capture target receptors (c‐Met and CD71) and a DNA logic assembly functioned as a computer processor to handle multiple inputs. As a result, the DNA assembly brings c‐Met and CD71 into close proximity, thus interfering with the ligand–receptor interactions of c‐Met and inhibiting its functions. Using this principle, a set of logic gates was created that respond to DNA strands or light irradiation, modulating the c‐Met/HGF signal pathways. This simple modular design provides a robust chemical tool for modulating cellular signal transduction.     

Light‐Induced Protein Dimerization by One‐ and Two‐Photon Activation of Gibberellic Acid Derivatives in Living Cells

We developed a highly efficient system for light‐induced protein dimerization in live cells using photo‐caged derivatives of the phytohormone gibberellic acid (GA₃). We demonstrate the application of the photo‐activatable chemical inducer of dimerization (CID) for the control of protein translocation with high spatiotemporal precision using light as an external trigger. Furthermore, we present a new two‐photon (2P)‐sensitive caging group, whose exceptionally high two‐photon cross section allows the use of infrared light to efficiently unleash the active GA₃ for inducing protein dimerization in living cells.     

Additive‐Free Green Light‐Induced Ligation Using BODIPY Triggers

Photochemical ligation is important in biomaterials engineering for spatiotemporal control of biochemical processes. Such reactions however generally require activation by high energy UV or short wavelength blue light, which can limit their use as a consequence of the potential of these high energy light sources to damage living cells. Herein, we present an additive‐free, biocompatible, chemical ligation triggered by mild visible light. BODIPY dyes with a pendant thioether attached at the meso‐position undergo photolysis of the [C?S] bond under green light (λ=530 nm) excitation, producing an ion pair intermediate that can react specifically with a propiolate group. The utility of this photochemical ligation in materials science is demonstrated by the fabrication of hydrogels with specific architectures, photo‐immobilization of biomacromolecules, and live cell encapsulation within a hydrogel scaffold.     

Mesomorphism,polymerization, and chirality induction in α‐cyanostilbene‐functionalized diacetylene‐assembled films: Photo‐triggered Z/E isomerization

Engineering of molecular stacking arrangement via environmental stimuli is of particular interest in stimuli‐responsive self‐assembling architectures. A novel dual photo‐functionalized diacetylene ((Z)‐CNBE‐DA) molecule was synthesized, in which photo‐responsive cyanostilbene moieties exhibited interesting Z‐E isomerization upon UV light irradiation and could be utilized to modulate mesomorphism, molecular stacking arrangement and resulting polymerization behavior. Rod‐like (Z)‐CNBE‐DA could self‐assemble into well‐defined lamellar structures and the helical polydiacetylene (PDA) chains could be formed upon irradiation with circularly polarized ultraviolet light (CPUL). However, the bent‐shaped (E)‐CNBE‐DA molecules only self‐assembled into irregular loose packing, inhibiting the formation of ordered helical PDA chains upon CPUL irradiation. In this work, we established the links between chemical structures, molecular packing engineering and photophysical properties, which would be of great fundamental value for the rational design of smart soft materials. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2458–2466     

Selection of DNA‐Encoded Small Molecule Libraries Against Unmodified and Non‐Immobilized Protein Targets

The selection of DNA‐encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand‐induced photo‐crosslinking, we show that iterated selections of DNA‐encoded libraries can be realized with unmodified and non‐immobilized protein targets.     

Sulfated Carbon Quantum Dots as Efficient Visible‐Light Switchable Acid Catalysts for Room‐Temperature Ring‐Opening Reactions

Acid catalytic processes play a classic and important role in modern organic synthesis. How well the acid can be controlled often plays the key role in the controllable synthesis of the products with high conversion yield and selectivity. The preparation of a novel, photo‐switchable solid‐acid catalyst based on carbon quantum dots is described. The carbon quantum dots are decorated with small amounts of hydrogensulfate groups and thus exhibit a photogenerated acidity that produces a highly efficient acid catalysis of the ring opening of epoxides with methanol and other primary alcohols. This reversible, light‐switchable acidity is shown to be due to photoexcitation and charge separation in the carbon quantum dots, which create an electron withdrawing effect from the acidic groups. The catalyst is easily separated by filtration, and we demonstrate multiple cycles of its recovery and reuse.     

A Reversible DNA Logic Gate Platform Operated by One‐ and Two‐Photon Excitations

We demonstrate the use of two different wavelength ranges of excitation light as inputs to remotely trigger the responses of the self‐assembled DNA devices (D‐OR). As an important feature of this device, the dependence of the readout fluorescent signals on the two external inputs, UV excitation for 1 min and/or near infrared irradiation (NIR) at 800 nm fs laser pulses, can mimic function of signal communication in OR logic gates. Their operations could be reset easily to its initial state. Furthermore, these DNA devices exhibit efficient cellular uptake, low cytotoxicity, and high bio‐stability in different cell lines. They are considered as the first example of a photo‐responsive DNA logic gate system, as well as a biocompatible, multi‐wavelength excited system in response to UV and NIR. This is an important step to explore the concept of photo‐responsive DNA‐based systems as versatile tools in DNA computing, display devices, optical communication, and biology.     

Photocaged Quinone Methide Crosslinkers for Light‐Controlled Chemical Crosslinking of Protein–Protein and Protein–DNA Complexes

Small‐molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo‐crosslinkers target almost all residues non‐specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)‐based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein–protein and protein–DNA networks and for structural biology.     

Liquid Crystal Networks on Thermoplastics: Reprogrammable Photo‐Responsive Actuators

Arbitrary shape (re)programming is appealing for fabricating untethered shape‐morphing photo‐actuators with intricate configurations and features. We present re‐programmable light‐responsive thermoplastic actuators with arbitrary initial shapes through spray‐coating of polyethylene terephthalate (PET) with an azobenzene‐doped light‐responsive liquid crystal network (LCN). The initial geometry of the actuator is controlled by thermally shaping and fixing the thermoplastic PET, allowing arbitrary shapes, including origami‐like folds and left‐ and right‐handed helicity within a single sample. The thermally fixed geometries can be reversibly actuated through light exposure, with fast, reversible area‐specific actuation such as winding, unwinding and unfolding. By shape re‐programming, the same sample can be re‐designed and light‐actuated again. The strategy presented here demonstrates easy fabrication of mechanically robust, recyclable, photo‐responsive actuators with highly tuneable geometries and actuation modes.     

Determination of genomic N3‐methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser‐induced fluorescence

Methylating substances alter DNA by forming N3‐methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF), we synthesized the N3mT‐3’‐phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N’‐nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5‐hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5‐methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE‐LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.     

Multi‐Light‐Responsive Quantum Dot Sensitized Hybrid Micromotors with Dual‐Mode Propulsion

CdS quantum dots/C₆₀ tubular micromotors with chemical/multi‐light‐controlled propulsion and “on‐the‐fly” acceleration capabilities are described. In situ growth of CdS quantum dots on the outer fullerene layer imparts this layer with light‐responsive properties in connection to inner Pt, Pd or MnO₂ layers. This is the first time that visible light is used to drive bubble‐propelled tubular micromotors. The micromotors exhibit a broad absorption range from 320 to 670 nm and can be wirelessly controlled by modulating light intensity and peroxide concentration. The built‐in accelerating optical system allows for the control of the velocity over the entire UV/Vis light spectra by modulating the catalyst surface chemistry. The light‐responsive properties have been also exploited to accelerate the chemical dealloying and propulsion of micromotors containing a Cu/Pd layer. Such dual operated hybrid micromotors hold considerable promise for designing smart micromachines for on‐demand operations, motion‐based sensing, and enhanced cargo transportation.     

On‐Tip Photo‐Modulated Molecular Printing

The concept of using cantilever‐free scanning probe arrays as structures that can modulate nanoscale ink flow and composition with light is introduced and evaluated. By utilizing polymer pen arrays with an opaque gold layer surrounding the base of the transparent polymer pyramids, we show that inks with photopolymerizable or isomerizable constituents can be used in conjunction with light channelled through the pyramids to control ink viscosity or composition in a dynamic manner. This on‐tip photo‐modulated molecular printing provides novel chemically and mechanically controlled approaches to regulating ink transport and composition in real time and could be useful not only for rapidly adjusting feature size but also for studying processes including photoreactions and mass transport at the nanoscale, self‐assembly, and cell–material interactions.     

Light‐Controlled Ion Transport through Biomimetic DNA‐Based Channels

Light‐controlled nanochannels are fabricated through self‐assembling azobenzene‐incorporated DNA (Azo‐DNA) strands to regulate ion transport. By switching between collapsed and relaxed states using visible and ultraviolet light alternately, the Azo‐DNA channels can be opened and closed because the conformation of Azo‐DNA changes, that is, Azo‐DNA is used as switchable controlling unit. In addition to sharing short response time and reversibility with other photoresponsive apparatuses, the Azo‐DNA‐based nanochannel system has advantages in good biocompatibility and versatile design, which could potentially be applied in light‐controlled drug release, optical information storage, and logic networks.     

Photo‐Tethers for the (Multi‐)Cyclic,Conformational Caging of Long Oligonucleotides

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne‐modified photolabile nucleosides into DNA sequences, followed by a Cu^I‐catalyzed alkyne–azide cycloaddition with bis‐azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full‐length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo‐tether strategy presented here provides a robust and versatile tool for the light‐activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.     

Photocatalysis Enables Visible‐Light Uncaging of Bioactive Molecules in Live Cells

The photo‐manipulation of bioactive molecules provides unique advantages due to the high temporal and spatial precision of light. The first visible‐light uncaging reaction by photocatalytic deboronative hydroxylation in live cells is now demonstrated. Using Fluorescein and Rhodamine derivatives as photocatalysts and ascorbates as reductants, transient hydrogen peroxides were generated from molecular oxygen to uncage phenol, alcohol, and amine functional groups on bioactive molecules in bacteria and mammalian cells, including neurons. This effective visible‐light uncaging reaction enabled the light‐inducible protein expression, the photo‐manipulation of membrane potentials, and the subcellular‐specific photo‐release of small molecules.