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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Enzyme-Artificial Enzyme Interactions as a Means for Discriminating among Structurally Similar Isozymes.

We describe the design and function of an artificial enzyme-linked receptor (ELR) that can bind different members of the glutathione-S-transferase (GST) enzyme family. The artificial enzyme-enzyme interactions distinctly affect the catalytic activity of the natural enzymes, the biomimetic, or both, enabling the system to discriminate among structurally similar GST isozymes.

Protein-Protein Communication and Enzyme Activation Mediated by a Synthetic Chemical Transducer.

The design and function of a synthetic "chemical transducer" that can generate an unnatural communication channel between two proteins is described. Specifically, we show how this transducer enables platelet-derived growth factor to trigger (in vitro) the catalytic activity of glutathione-s-transferase (GST), which is not its natural enzyme partner. GST activity can be further controlled by adding specific oligonucleotides that switch the enzymatic reaction on and off. We also demonstrate that a molecular machine, which can regulate the function of an enzyme, could be used to change the way a prodrug is activated in a "programmable" manner.

Sensing Protein Surfaces with Targeted Fluorescent Receptors.

A methodology for creating fluorescent molecular sensors that respond to changes that occur on the surfaces of specific proteins is presented. This approach, which relies on binding cooperatively between a specific His-tag binder and a nonspecific protein-surface receptor, enabled the development of a sensor that can track changes on the surface of a His-tag-labeled calmodulin (His-CaM) upon interacting with metal ions, small molecules, and protein binding partners. The way this approach was used to detect dephosphorylation of an unlabeled calmodulin-dependent protein kinase II (CaMKII), and the binding of Bax BH3 to His-tagged B-cell lymphoma 2 (Bcl-2) protein is also presented.

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.

Targeted protein surface sensors as a tool for analyzing small populations of proteins in biological mixtures.

Optical cross-reactive sensor arrays (the so-called chemical "noses/tongues") have recently been demonstrated as a powerful tool for high-throughput protein detecting and analysis. Nevertheless, applying this technology to biomarker detection is complicated by the difficulty of non-selective sensors to operate in biological mixtures. Herein we demonstrate a step toward circumventing this limitation by using self-assembled fluorescent receptors consisting of two distinct recognition motifs: specific and non-specific. When combined in an array, binding cooperatively between the specific and non-specific protein binders enables the system to discriminate among closely related isoform biomarkers even in the presence of serum proteins or within human urine.

Unnatural enzyme activation by a metal-responsive regulatory protein.

As a result of calcium ion binding, the calcium-dependent regulatory protein calmodulin (CaM) undergoes a conformational change, enabling it to bind to and activate a variety of enzymes. However, the detoxification enzyme glutathione S-transferase (GST) is notably not among the enzymes activated by CaM. In this study, we demonstrate the feasibility of establishing, in vitro, an artificial regulatory link between CaM and GST using bifunctional chemical transducer (CT) molecules possessing binders for CaM and GST. We show that the CTs convert the constitutively active GST into a triggerable enzyme whose activity is unnaturally regulated by the CaM conformational state and consequently, by the level of calcium ions. The ability to reconfigure the regulatory function of CaM demonstrates a novel mode by which CTs could be employed to mediate artificial protein crosstalk, as well as a new means to achieve artificial control of enzyme activity by modulating the coordination of metal ions. Within this study, we also investigated the impact of covalent interaction between the CTs and the enzyme target. This investigation offers further insights into the mechanisms governing the function of CTs and the possibility of rendering them isoform specific.

Authorizing multiple chemical passwords by a combinatorial molecular keypad lock.

A combinatorial fluorescent molecular sensor operates as a highly efficient molecular security system. The ability of a pattern-generating molecule to process diverse sets of chemical inputs, discriminate among their concentrations, and form multivalent and kinetically stable complexes is demonstrated as a powerful tool for processing a wide range of chemical "passwords" of different lengths. This system thus indicates the potential for obtaining unbreakable combination locks at the molecular scale.

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.

Molecule and electron transfer through coordination-based molecular assemblies.

This study provides insight into the internal structure of surface-confined molecular assemblies. The permeability of the layer-by-layer grown thin films can be controlled systematically by varying their composition and the structure of their molecular components. Moreover, the thickness can be used to control molecule permeation versus electron transfer.