Publications

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.

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 cells 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 cells environment and consequent protein expression profile.

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.

Conspectus Fluorescent molecular sensors, often referred to as \u201cturn-on\u201d or \u201cturn-off\u201d 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 Aβ 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.

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.

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.

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.

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.

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.

A method for decorating bacteria with synthetic receptors while they are attached to a solid support is described. This approach involves two steps. First, E. coli expressing a Histagged outer membrane protein C (HisOmpC) are attached to a monolayer of DNA duplexes appended with a trinitrilotriacetic acid (triNTA) group. The remaining Histags, which are not involved in the interaction with the monolayer, can then be modified with additional triNTAappended duplexes that bear different functionalities. By labelling the surfacebound bacteria with an oligodeoxynucleotide (ODN) appended with a fluorescent dye and by turning off its emission using a complementary, quencherbearing strand, we demonstrate the versatility of this approach, as well as a means to modify cell membrane proteins on a solid support using simple selfassembly processes.

Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in liveEscherichia 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.

Labeling of proteins with small-molecule-based fluorescent probes provides a powerful tool to determine their expression level and localization in living cells. We propose a method for increasing and stabilizing the emission of such probes using supramolecular hosts such as β-cyclodextrin (β-CD). Although the emission of a His-tag binding probe was enhanced in the presence of β-CD, binding of the probe-β-CD complex to a His-tagged protein led to partial β-CD displacement and consequently, a reduction in the emission signal.

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 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.

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.

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 (β-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 β-CD as an "antidote" that prevents GST inhibition. The relevance of this system to biomedical applications is demonstrated by using the INHIBIT gate and β-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 β-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 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.

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.

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.

Different amyloid beta (A beta) 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 A beta aggregation states, which result from the formation of low molecular weight oligomers, high molecular weight oligomers, protofibrils, and fibrils.

Protein surface recognition by fluorescent molecular sensors poses an immense challenge in supramolecular recognition chemistry owing to the immense difficulty of selectively targeting these large, relatively flat and non-contiguous domains. The fact that protein surfaces can exhibit different charges, topologies, and posttranslational modifications that can be found in other proteins in the mixture is an additional factor that complicates targeting and therefore, sensing specific protein surface modifications. A recent report, however, shows that the difficulty of sensing changes that occur on the surface of specific proteins could be circumvented by attaching a relatively non-specific synthetic receptor to a specific protein binder. The latter brings the receptor near the target protein and enhances its affinity toward its surface. Modifying the synthetic receptor with an environmentally sensitive fluorescent reporter along with suitable recognition elements enables such systems to target specific regions on protein surfaces and consequently, track modifications that result from conformational changes or binding interactions.

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 cells 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.

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.

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.

The most basic functions of the cell such as proliferation, differentiation, metabolism and apoptosis are dependent on signal transduction pathways that determine the response of the cell to various environmental signals [1]. These pathways generally begin by the binding of a ligand to an extracellular receptor, which triggers an intracellular signal cascade. The propagation of the signal within the cell is mediated by various proteinprotein interactions, in which enzymes activate or deactivate one another with high specificity. Because numerous diseases such as cancer are associated with dysregulation of these networks, much effort has been devoted to developing drugs that disrupt proteinprotein interactions or inhibit enzymes that are involved in abnormal cell signaling pathways [2,3]. Herceptin and gleevec [4], for example, are well-known anticancer drugs that inhibit the signaling pathways of receptor tyrosine kinases (RTKs) in different ways. The first is an antibody-based drug that binds the extracellular domain of HER-2, a member of the EGFRs family. It disrupts dimerization of HER-2 with neighboring HER receptors and consequently prevents the activation of intracellular kinases. The second drug is a cell-permeable molecule that directly inhibits the intracellular kinase of the PDGF receptor as well as the activity of ABL or c-kit kinases. Despite their different structures and mechanisms of action, treatment with hereceptin or gleevec eventually results in kinase inhibition, which shuts down an intracellular communication channel that is critical to the survival of the cancer cell. These drugs therefore demonstrate well the effectiveness of signal transduction therapy of cancer [3] that exploits the dependence of cancer cells on fewer, upregulated signaling pathways. In contrast to healthy cells that can minimize the effect of these drugs by activating alternative signaling networks, cancer cells cannot evade this acute interference in their molecular circuitry, which leads to selective cell death [3].

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.

We show that the conversion of a known intercalating dye (i.e., thiazole orange) into a bivalent protein binder could lead to the realization of a novel class of 'turn-on' fluorescent molecular probes that detect proteins with high affinity, selectivity, and a high signal-to-noise (S/N) ratio. The feasibility of the approach is demonstrated with monomolecular probes that light-up in the presence of three different proteins: acetylcholinesterase (AChE), glutathione-s-transferase (GST), or avidin (Av) at low concentrations and with minimal background signal. The way by which such probes can be used to detect individual protein isoforms and be applied in inhibitor screening, cell imaging, and biomarker detection is described.

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.

Summary srGAP proteins regulate cell migration and morphogenesis by shaping the structure and dynamics of the cytoskeleton and membranes. First discovered as intracellular effectors for the Robo1 axon-guidance receptor, srGAPs were later identified as interacting with several other nuclear and cytoplasmic proteins. In all these cases, the srGAP SH3 domain mediates protein-protein interactions by recognizing a short proline-rich segment on the cognate-binding partner. However, as interactions between the isolated SH3 domain and a selected set of ligands show weak affinity and low specificity, it is not clear how srGAPs are precisely recruited to their signaling sites. Here, we report a two-component molecular mechanism that regulates ligand binding to srGAP2 by on the one hand dramatically tightening their association and on the other, moderately autoinhibiting and restricting binding. Our results allow the design of point mutations for better probing of srGAP2 activities, and may facilitate the identification of new srGAP2 ligands.

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.

Combinatorial fluorescent molecular sensors constitute a unique class of analytical systems that integrate the properties of small-molecule luminescent sensors and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, these sensors can differentiate between a wide range of analyte combinations and concentrations, akin to pattern-generating arrays. On the other hand, they can operate in the microscopic world, which macroscopic analytical devices cannot access. This feature article summarizes the different approaches that can be used for obtaining multianalyte detection by fluorescent molecular sensors. In particular, it highlights an exciting prospect in the analytical sciences: differential sensing at the molecular level.

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.

Working together to uncover the truth: A molecule-sized diagnostic system combining several recognition elements and four fluorescence-emission channels enabled the identification of a wide range of pharmaceuticals on the basis of distinct photophysical processes. The molecular sensor (see simplified representation; ID=identification) was also used to analyze drug concentrations and combinations in urine samples in a high-throughput manner.

Current approaches to medical diagnostics and drug design are largely based on the ability of monoclonal antibodies or synthetic molecules to bind proteins with high affinity and selectivity. In recent years, however, an alternative approach to protein recognition has emerged, in which proteins are identified using non-specific receptor arrays that are inspired by the olfactory neural system. An ultimate challenge for such systems is realizing a single, high-throughput analytical device that can effectively diagnose a range of medicinally relevant proteins. Such devices might overcome the difficulties associated with designing potent synthetic receptors for proteins and hence, could open up new possibilities in medical diagnostics, pathogen detection, and proteomics. Here we summarize recent developments in this area and also highlight its limitations and the challenges that this exciting interdisciplinary field faces. In particular, the goal of this review is to underscore the basic parameters required for obtaining combinatorial sensors for proteins and more importantly, to elucidate the rational methodologies that can be applied for systematically improving these promising analytical devices.

(Figure Presented) Here we show how different principles developed in the area of molecular logic gates can be applied to diagnostic technologies for proteins. Simultaneous operation of YES NOT and PASS 1 logic gates, produced by a protein sensing ensemble of DNA G-quadruplexes, is used to encode concentration levels of medicinally important proteins. An AND logic gate is another example, where molecular computation can be used to follow the interaction between proteins and metal ions. Combination of molecular Boolean logic with combinatorial sensing is demonstrated as a general strategy to realizing small scale, real time diagnosis of a variety of protein samples.

Sniffing out proteins: Fluorescent DNA G-quadruplexes have been used for building versatile signaling receptors for proteins in a single solution. Introducing a protein sample to the ensemble results in a unique emission signature for unambiguous identification (see scheme, R=fluorophore). The self-assembled, pattern-based protein detection systems are easily fabricated, have the potential for high-throughput operations, and have the ability to handle small protein samples.

This paper describes a new concept in the way information can be protected at the molecular scale. By harnessing the principles of molecular Boolean logic, we have designed a molecular device that mimics the operation of an electronic keypad lock, e.g., a common security circuit used for numerous applications, in which access to an object or data is to be restricted to a limited number of persons. What distinguishes this lock from a simple molecular logic gate is the fact that its output signals are dependent not only on the proper combination of the inputs but also on the correct order by which these inputs are introduced. In other words, one needs to know the exact passwords that open this lock. The different password entries are coded by a combination of two chemical and one optical input signals, which can activate, separately, blue or green fluorescence output channels from pyrene or fluorescein fluorophores. The information in each channel is a single-bit light output signal that can be used to authorize a user, to verify authentication of a product, or to initiate a higher process. This development not only opens the way for a new class of molecular decision-making devices but also adds a new dimension of protection to existing defense technologies, such as cryptography and steganography, previously achieved with molecules.

Over the past decade, there has been remarkable progress in the development of molecular logic and arithmetic systems, which has brought chemists closer to the realization of a molecular scale calculator (a Moleculator). This paper describes a significant step in this direction. By integrating past and new approaches for molecular logic reconfiguration, we were able to load advanced arithmetic calculations onto a single molecular species. Exchanging chemical inputs, monitoring at several wavelengths simultaneously, as well as using negative logic for the transmittance mode significantly increase the input and output information channels of the processing molecule. Changing the initial state of the processor is an additional approach used for altering the logical output of the device. Finally, introducing degeneracy to the chemical inputs or, alternatively, controlling their interactions to form identical chemical states minimizes the complexity of realizing three-bits addition and subtraction at the molecular scale. Consequently, using a commercially available fluorescein molecule, acid and base chemical inputs, and a simple UV-vis measurement setup, integration of a full-adder and, for the first time, a full-subtractor is now possible within individual molecules.

A highly efficient and simple molecular arithmetic system based on a plain fluorescene dye, capable of performing a full scale of elementary addition and subtraction algebraic operations is reported. For achieving boolean functions through scid-base interactions and their control over multistate molecular switches are described. A half-adder, performing a+b algebraic operations, is obtained with two identical sodium hydroxide solutions as inputs and by monitoring the outputs at 447 and 501 nm. Subtraction is achieved by operating the half-subtractor first using acid and then using base as input numbers. This molecular arithmetic unit demonstrates a general applicable method for resetting of chemical computation system.

We describe the design and function of a molecular logic system, by which a combinatorial recognition of the input signals is utilized to efficiently process chemically encoded information. Each chemical input can target simultaneously multiple domains on the same molecular platform, resulting in a unique combination of chemical states, each with its characteristic fluorescence output. Simple alteration of the input reagents changes the emitted logic pattern and enables it to perform different algebraic operations between two bits, solely in the fluorescence mode. This system exhibits parallelism in both its chemical inputs and light outputs.

The multifunctional nature of proteins that have iron-heme cofactors with noncovalent histidine linkage to the protein is controlled by the heme environment. Previous studies of these active-site structures show that the primary difference is the length of the iron-proximal histidine bond, which can be controlled by the degree of H-bonding to this histidine. Great efforts to mimic these functions with synthetic analogues have been made for more than two decades. The peroxidase models resulted in several catalytic systems capable of a large range of oxidative transformations. Most of these model systems modified the porphyrin ring covalently by directly binding auxiliary elements that control and facilitate reactivity; for example, electron- donating or -withdrawing substituents. A biomimetic approach to enzyme mimicking would have taken a different route, by attempting to keep the porphyrin ring system unaltered, as close as possible to its native form, and introducing all modifications at or close to the axial coordination sites. Such a model system would be less demanding synthetically, would make it easy to study the effect of a single structural modification, and might even provide a way to probe effects resulting from porphyrin exchange. We introduce here an alternative model system based on these principles. It consists of a two component system: a bis-imidazolyl ligand and an iron-porphyrin (readily substituted by a hemin). All modifications were introduced only to the ligand that engulfs the porphyrin and binds to the iron's fifth and sixth coordination sites. We describe the design, synthesis, and characterization of nine different model compounds with increased complexity. The primary tool for characterizing the environment of each complex Fe¹¹¹ center was the Extended X-ray Absorption Fine Structure (EXAFS) measurements, supported by UV/Vis, IR, and NMR spectroscopy and by molecular modeling. Introduction of asymmetry, by attaching different imidazoles as head groups, led to the formation of two axial bonds of different length. Addition of H-bonds to one of the imidazoles in an advanced model increased this differentiation and expanded the porphyrin ring. These complexes were found to be almost identical in structure to peroxidase active sites. Similarly to the peroxidases and other synthetic models, these compounds stabilize the green, compound I-like intermediate, and catalyze the oxidation of organic substrates.