Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

Fluorescent Investigation of Proteins Using DNA-Synthetic Ligand Conjugates.

The unfathomable role that fluorescence detection plays in the life sciences has prompted the development of countless fluorescent labels, sensors, and analytical techniques that can be used to detect and image proteins or investigate their properties. Motivated by the demand for simple-to-produce, modular, and versatile fluorescent tools to study proteins, many research groups have harnessed the advantages of oligodeoxynucleotides (ODNs) for scaffolding such probes. Tight control over the valency and position of protein binders and fluorescent dyes decorating the polynucleotide chain and the ability to predict molecular architectures through self-assembly, inherent solubility, and stability are, in a nutshell, the important properties of DNA probes. This paper reviews the progress in developing DNA-based, fluorescent sensors or labels that navigate toward their protein targets through small-molecule (SM) or peptide ligands. By describing the design, operating principles, and applications of such systems, we aim to highlight the versatility and modularity of this approach and the ability to use ODN-SM or ODN-peptide conjugates for various applications such as protein modification, labeling, and imaging, as well as for biomarker detection, protein surface characterization, and the investigation of multivalency.

Chemically programmable bacterial probes for the recognition of cell surface proteins.

Common methods to label cell surface proteins (CSPs) involve the use of fluorescently modified antibodies (Abs) or small-molecule-based ligands. However, optimizing the labeling efficiency of such systems, for example, by modifying them with additional fluorophores or recognition elements, is challenging. Herein we show that effective labeling of CSPs overexpressed in cancer cells and tissues can be obtained with fluorescent probes based on chemically modified bacteria. The bacterial probes (B-probes) are generated by non-covalently linking a bacterial membrane protein to DNA duplexes appended with fluorophores and small-molecule binders of CSPs overexpressed in cancer cells. We show that B-probes are exceptionally simple to prepare and modify because they are generated from self-assembled and easily synthesized components, such as self-replicating bacterial scaffolds and DNA constructs that can be readily appended, at well-defined positions, with various types of dyes and CSP binders. This structural programmability enabled us to create B-probes that can label different types of cancer cells with distinct colors, as well as generate very bright B-probes in which the multiple dyes are spatially separated along the DNA scaffold to avoid self-quenching. This enhancement in the emission signal enabled us to label the cancer cells with greater sensitivity and follow the internalization of the B-probes into these cells. The potential to apply the design principles underlying B-probes in therapy or inhibitor screening is also discussed here.

Molecules that Generate Fingerprints: A New Class of Fluorescent Sensors for Chemical Biology, Medical Diagnosis, and Cryptography.

Fluorescent molecular sensors, often referred to as "turn-on" or "turn-off" fluorescent probes, are synthetic agents that change their fluorescence signal in response to analyte binding. Although these sensors have become powerful analytical tools in a wide range of research fields, they are generally limited to detecting only one or a few analytes. Pattern-generating fluorescent probes, which can generate unique identification (ID) fingerprints for different analytes, have recently emerged as a new class of luminescent sensors that can address this limitation. A unique characteristic of these probes, termed ID-probes, is that they integrate the qualities of conventional small-molecule-based fluorescent sensors and cross-reactive sensor arrays (often referred to as chemical, optical, or electronic noses/tongues). On the one hand, ID-probes can discriminate between various analytes and their combinations, akin to array-based analytical devices. On the other hand, their minute size enables them to analyze small-volume samples, track dynamic changes in a single solution, and operate in the microscopic world, which the macroscopic arrays cannot access.Here, we describe the principles underlying the ID-probe technology, as well as provide an overview of different ID-probes that have been developed to date and the ways they can be applied to a wide range of research fields. We describe, for example, ID-probes that can identify combinations of protein biomarkers in biofluids and in living cells, screen for several protein inhibitors simultaneously, analyze the content of Aß aggregates, as well as ensure the quality of small-molecule and biological drugs. These examples highlight the relevance of this technology to medical diagnosis, bioassay development, cell and chemical biology, and pharmaceutical quality assurance, among others. ID-probes that can authorize users and protect secret data are also presented and the mechanisms that enable them to hide (steganography), encrypt (cryptography), and prevent access to (password protection) information are discussed.The versatility of this technology is further demonstrated by describing two types of probes: unimolecular ID-probes and self-assembled ID-probes. Probes from the first type can operate inside living cells, be recycled, and their initial patterns can be more easily obtained in a reproducible manner. The second type of probes can be readily modified and optimized, allowing one to prepare various different probes from a much wider range of fluorescent reporters and supramolecular recognition elements. Taken together, these developments indicate that the ID-probe sensing methodology is generally applicable, and that such probes can better characterize analyte mixtures or process chemically encoded information than can the conventional fluorescent molecular sensors. We therefore hope that this review will inspire the development of new types of pattern-generating probes, which would extend the fluorescence molecular toolbox currently used in the analytical sciences.

Applications of Bacteria Decorated with Synthetic DNA Constructs.

The advent of DNA nanotechnology has revolutionized the way DNA has been perceived. Rather than considering it as the genetic material alone, DNA has emerged as a versatile synthetic scaffold that can be used to create a variety of molecular architectures. Modifying such self-assembled structures with bio-molecular recognition elements has further expanded the scope of DNA nanotechnology, opening up avenues for using synthetic DNA assemblies to sense or regulate biological molecules. Recent advancements in this field have lead to the creation of DNA structures that can be used to modify bacterial cell surfaces and endow the bacteria with new properties. This mini-review focuses on the ways by which synthetic modification of bacterial cell surfaces with DNA constructs can expand the natural functions of bacteria, enabling their potential use in various fields such as material engineering, bio-sensing, and therapy. The challenges and prospects for future advancements in this field are also discussed.

An Optical Probe for Real-Time Monitoring of Self-Replicator Emergence and Distinguishing between Replicators.

Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, we now developed a pattern-generating fluorescent molecular probe (an ID-probe) capable of discriminating replicators of different chemical composition and monitoring the process of replicator formation in real time, giving distinct signatures for starting materials, intermediates, and final products. Optical monitoring of replicators dramatically reduces the analysis time and sample quantities compared to most currently used methods and opens the door for future high-throughput experimentation in protocell environments.

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions.

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.

Assessing changes in the expression levels of cell surface proteins with a turn-on fluorescent molecular probe.

Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in live Escherichia coli. One of these probes was designed to light up upon binding, which provided the means to assess changes in the His-OmpC expression levels by taking a simple fluorescence spectrum.

Encrypting messages with artificial bacterial receptors.

A method for encrypting messages using engineered bacteria and different fluorescently labeled synthetic receptors is described. We show that the binding of DNA-based artificial receptors to E. coli expressing His-tagged outer membrane protein C (His-OmpC) induces a Förster resonance energy transfer (FRET) between the dyes, which results in the generation of a unique fluorescence fingerprint. Because the bacteria continuously divide, the emission pattern generated by the modified bacteria dynamically changes, enabling the system to produce encryption keys that change with time. Thus, this development indicates the potential contribution of live-cell-based encryption systems to the emerging area of information protection at the molecular level.

Glycoform Differentiation by a Targeted, Self-Assembled, Pattern-Generating Protein Surface Sensor.

A method for generating targeted, pattern-generating, protein surface sensors via the self-assembly of modified oligodeoxynucleotides (ODNs) is described. The simplicity by which these systems can be created enabled the development of a sensor that can straightforwardly discriminate between distinct glycoform populations. By using this sensor to identify glycosylation states of a therapeutic protein, we demonstrate the diagnostic potential of this approach as well as the feasibility of integrating a wealth of supramolecular receptors and sensors into higher-order molecular analytical devices with advanced properties. For example, the facile device integration was used to attach the well-known anthracene-boronic acid (An-BA) probe to a biomimetic DNA scaffold and consequently, to use the unique photophysical properties of An-BA to improve glycoform differentiation. In addition, the noncovalent assembly enabled us to modify the sensor with a trinitrilotriacetic acid (tri-NTA)-Ni2+ complex, which endows it with selectivity toward a hexa-histidine tag (His-tag). The selective responses of the system to diverse His-tag-labeled proteins further demonstrate the potential applicability of such sensors and validate the mechanism underlying their function.

Decorating bacteria with self-assembled synthetic receptors.

The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs. We show that the local concentration of these receptors on the bacterial membrane and their structure can be reversibly controlled using suitable chemical signals, in a way that resembles changes that occur with CSP expression levels or posttranslational modifications (PTMs), respectively. We also show that these modifications can endow the bacteria with programmable properties, akin to the way CSP responses can induce cellular functions. By programming the bacteria to glow, adhere to surfaces, or interact with proteins or mammalian cells, we demonstrate the potential to tailor such biomimetic systems for specific applications.

Molecular Logic as a Means to Assess Therapeutic Antidotes.

An emerging direction in the area of molecular logic and computation is developing molecular-scale devices that can operate in complex biological environments, such as within living cells, which are beyond the reach of conventional electronic devices. Herein we demonstrate, at the proof-of-principle level, how concepts applied in the field of molecular logic gates can be used to convert a simple fluorescent switch (YES gate), which lights up in the presence of glutathione s-transferase (GST), into a medicinally relevant INHIBIT gate that responds to both GST and beta-cyclodextrin (ß-CD) as input signals. We show that the optical responses generated by this device indicate the ability to use it as an enzyme inhibitor, and more importantly, the ability to use ß-CD as an "antidote" that prevents GST inhibition. The relevance of this system to biomedical applications is demonstrated by using the INHIBIT gate and ß-CD to regulate the growth of breast cancer cells, highlighting the possibility of applying supramolecular inputs, commonly used to control the fluorescence of molecular logic gates, as antidotes that reverse the toxic effect of chemotherapy agents. We also show that the effect of ß-CD can be prevented by introducing 1-adamantanecarboxylic acid (Ad-COOH) as an additional input signal, indicating the potential of obtaining precise, temporal control over enzyme activity and anticancer drug function.

A Molecular Secret Sharing Scheme.

A method for implementing a secret sharing scheme at the molecular level is presented. By creating molecular code generators that are self-assembled from several molecular components, we established a means for distributing distinct code-activating elements among several participants. In this way, an authorization code can only be generated when all the participants are present, which ensures that highly secured systems cannot be operated by unauthorized individuals or disloyal users. Additional layers of protection result from the ability to program the security code by replacing one or several molecular components and by subjecting the system to distinct chemical inputs.

Protein recognition by a pattern-generating fluorescent molecular probe.

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

User Authorization at the Molecular Scale.

Electronic user authorization systems help us maintain our privacy in many aspects of everyday life. However, the increasing difficulty to secure access and/or information digitally has inspired chemists to devise alternative, molecular approaches, in which users are identified by chemical means. The potential advantages of using molecular user authentication systems over conventional electronic devices are their versatility and unusual operating principles, which complicate replicating and, consequently, breaking into molecular security devices. Their molecular scale is another unique property that enables hiding such systems and, consequently, applying steganography as an additional layer of protection. Although the area of molecular-based user authorization is still in its infancy, the development of various molecular keypad locks and, more recently, a password-protected molecular cryptographic machine, indicate the possibility of protecting information at the molecular scale.

Analyzing Amyloid Beta Aggregates with a Combinatorial Fluorescent Molecular Sensor.

Different amyloid beta (Aß) aggregates can be discriminated by a combinatorial fluorescent molecular sensor. The unique optical fingerprints generated by the unimolecular analytical device provide a simple means to differentiate among aggregates generated from different alloforms or through distinct pathways. The sensor has also been used to track dynamic changes that occur in Aß aggregation states, which result from the formation of low molecular weight oligomers, high molecular weight oligomers, protofibrils, and fibrils.

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy.

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

Message in a molecule.

Since ancient times, steganography, the art of concealing information, has largely relied on secret inks as a tool for hiding messages. However, as the methods for detecting these inks improved, the use of simple and accessible chemicals as a means to secure communication was practically abolished. Here, we describe a method that enables one to conceal multiple different messages within the emission spectra of a unimolecular fluorescent sensor. Similar to secret inks, this molecular-scale messaging sensor (m-SMS) can be hidden on regular paper and the messages can be encoded or decoded within seconds using common chemicals, including commercial ingredients that can be obtained in grocery stores or pharmacies. Unlike with invisible inks, however, uncovering these messages by an unauthorized user is almost impossible because they are protected by three different defence mechanisms: steganography, cryptography and by entering a password, which are used to hide, encrypt or prevent access to the information, respectively.

Sensing Protein Surfaces with Targeted Fluorescent Receptors.

Invited for the cover of this issue is the group of David Margulies at the Weizmann Institute of Science (Israel). The image highlights the analogy between fluorescent molecular sensors and a miniaturized camera that can capture changes that occur at the nanoscale and shed light on the structural state of proteins. Read the full text of the article at 10.1002/chem.201502069.