Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.Keywords: BRET; creatinine; LUMABS; NanoLuc; protein engineering; sensor;

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.Keywords: BRET; caspase sensor; mNeonGreen; NanoLuc; plate reader assay; single-cell imaging;

Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.

DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control.

Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn2+ sensor into a light-activatable Zn2+ binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn2+ between two Zn2+ binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn2+ affinity upon illumination, which is fully reversible following dark adaptation. The Zn2+ affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn2+ binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn2+ levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.Keywords: fluorescent sensor; light switching; metal binding; optogenetics; protein engineering; Vivid

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time.

The Zn2+-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn2+ from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn2+ release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn2+ concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn2+, eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn2+ and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn2+ in both cytosol and ER, suggesting that Zn2+ was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn2+ levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn2+, nor in experiments in which cytosolic and ER Zn2+ were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF–ionomycin treatment does not result in significant changes in cytosolic Zn2+ levels as a result from Zn2+ release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn2+ dyes.

Förster resonance energy transfer (FRET) sensors and other ratiometric probes are increasingly used in life sciences to obtain quantitative information in complex environments such as the cell interior or blood plasma. When using FRET sensors, either to determine the affinity of the sensor for its analyte in vitro, or to apply the sensor to measure an unknown concentration in situ, the ratio of donor and acceptor emission is commonly used as a measure of the sensor occupancy. However, it has been recently demonstrated that the underlying assumption of a linear relationship between the emission ratio and the relative sensor occupancy is not correct. Here we present a simple solution to this problem by using the fluorescence intensity at the isosbestic wavelength as an internal standard. The isosbestic wavelength of FRET-sensors based on the widely used CFP/YFP pair was determined to be at 513 nm, independent of the specific sensor architecture and pH. We show that using the ratio of either the donor or the acceptor emission and the emission at the isosbestic wavelength of 513 nm provides a straightforward method to obtain accurate Kd values from in vitro titration experiments. In addition, using two recently developed FRET sensors for Zn2+ we show that the same approach can be used to allow more accurate quantification in live cell imaging experiments. We believe that this approach provides a generic solution to retain the advantages of ratiometric measurements, without compromising on analytical accuracy.Keywords: BRET sensor; FRET sensor; intracellular imaging; isosbestic point; Kd determination; ratiometric sensors; Zn2+ sensors

DNA-templated reversible assembly of an enzyme–inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-β-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template strand. Formation of a rigid dsDNA linker upon addition of a complementary target strand disrupts the enzyme–inhibitor complex and results in the restoration of enzyme activity, enabling detection of as little as 2 fmol DNA. The noncovalent assembly of the complex allows easy tuning of target and template strands without changing the oligonucleotide-functionalized enzyme and inhibitor domains. Using a panel of eight different template sequences, restoration of enzyme activity was only observed in the presence of the target viral DNA sequence. The use of stable, well-characterized protein domains and the intrinsic modularity of our system should allow easy integration with DNA/RNA-based logic circuits for applications in biomedicine and molecular diagnostics.Keywords: bionanotechnology; molecular switch; reporter enzyme; self-assembly; β-lactamase;

Zn2+ plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn2+ homeostasis and the putative signaling role of Zn2+, various fluorescent sensors have been developed that allow monitoring of Zn2+ concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn2+ sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn2+, but distinctly different concentrations have been reported when using these sensors to measure Zn2+ concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn2+ with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn2+ in the cytosol but was also successfully used for measuring Zn2+ in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn2+ simultaneously in the cytosol and the ER or mitochondria.

Genetically-encoded fluorescent sensor proteins are attractive tools for studying intracellular Zn2+ homeostasis and signaling. Here we provide an overview of recently developed sensors based on Förster Resonance Energy Transfer (FRET). The pros and cons of the various sensors are discussed with respect to Zn2+ affinity, dynamic range, intracellular targeting and multicolor imaging. Recent applications of these sensors are described, as well as some of the challenges that remain to be addressed in future research.

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)−linker−enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (ΔGo–c0 = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange–linker–mCherry fusion proteins yielded a toolbox of new mOrange–mCherry combinations that allowed tuning of their intramolecular interaction from very weak (ΔGo–c0 = −0.39 kCal/mol) to relatively stable (ΔGo–c0 = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean–Citrine pair by mOrange–mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies.

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1:1 antibody–ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide–dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide–dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.

Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an intramolecular interaction between TEM1 β-lactamase and its inhibitor protein BLIP. Using the HIV1-p17 antibody as an initial target, the interaction between enzyme and inhibitor was carefully tuned to yield a reporter enzyme whose activity increased 10-fold in the presence of pM antibody concentrations. Reporter enzymes for two other antibodies (HA-tag and Dengue virus type I) were obtained by simply replacing the epitope sequences. This new sensor design represents a modular and generic approach to construct antibody reporter enzymes without the cumbersome optimization required by previous engineering strategies.

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn2+ sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn2+ FRET sensors to image Zn2+ over a broad concentration range in the same cellular compartment.

We previously reported the development of high affinity Zn2+ FRET sensors based on the Zn2+-mediated interaction between the CXXC motifs present in the copper chaperone proteins ATOX1 and WD4. By systematically substituting several of these cysteines for methionines, we constructed sensor variants that retain a high affinity for Cu+, while effectively abolishing their ability to form stable tetrahedral Zn2+ complexes.

Detection of antibodies is essential for the diagnosis of many disease states, including infectious diseases, autoimmune diseases and allergies. Most current antibody detection assays involve multistep detection schemes in which molecular recognition and signal generation are separate processes. A well-known example is the enzyme-linked immunosorbent assay (ELISA), which combines high sensitivity and specificity with strong signal amplification. However, ELISA and other heterogeneous methods require multiple, time-consuming washing and incubation steps, which limits their applicability in point-of-care diagnostics and high-throughput applications. In recent years, several new antibody detection strategies have been developed in which antibody binding and signal generation are integrated within a single biomolecular reporter. These strategies aim to rival ELISA in terms of sensitivity and specificity, while decreasing the time and effort required to perform an assay. Here, we review recent developments in this field according to their mechanism of action and discuss their advantages and limitations.

A modular self-assembly strategy is presented that allows the non-covalent synthesis of multivalent protein dendrimers using the strong interaction between choline-functionalized dendrimers and the choline binding protein C-LytA. Choline dendrimers displaying fusion proteins of C-LytA and the collagen binding protein CNA35 represent attractive multivalent targeting ligands for collagen imaging.

A protease-activatable collagen targeting probe (proCNA35) is synthesized by conjugation of a synthetic collagen fragment to the collagen binding protein CNA35via a protease-cleavable linker. Cleavage of the linker by MMP1 releases the intramolecular inhibition of the collagen binding site and restores its collagen binding capacity.

Introduction of a (Cys)4 metal binding site at the dimerization interface of two fluorescent protein domains yields a chelating FRET sensor protein that shows a 2500-fold selectivity for Cd2+ over Zn2+ by taking advantage of their different ionic radii.

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB2) and tetravalent (AB4) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB4 system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.Strength in numbers: semi-synthetic multivalent collagen binding proteins were obtained by native chemical ligation of CNA35-thioesters and cysteine-functionalized divalent (AB2) and tetravalent (AB4) dendritic wedges.

Protein-functionalized micelles and liposomes are attractive delivery systems for applications ranging from targeted drug delivery to molecular imaging. In particular, systems that use pegylated phospholipids have become popular, but little is known about the stability of these lipid-functionalized proteins toward exchange. In this study, Förster resonance energy transfer (FRET) between the fluorescent proteins ECFP and EYFP was used to investigate the lipid exchange behavior of protein-functionalized liposomes and micelles. Native chemical ligation was used as an efficient method to site-specifically couple varying amounts of proteins to pegylated phospholipids. No exchange was observed between protein-functionalized phospholipids in sterically stabilized liposomes. In micelles, however, protein-functionalized lipids were found to exchange with a half-time of exchange ranging from almost 2 h at room temperature to 4 min at 37 °C. These pegylated micelles remained intact at lipid concentrations down to 0.15 μM, indicating that they are even more stable than previously assumed. The results obtained in this study provide a useful frame of reference for assessing the potential role of protein exchange in biomedical applications of these lipid-based nanoparticles.

Cytochrome c′ from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN− to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c′ and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV–vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.

Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.

A protease-activatable collagen targeting probe (proCNA35) is synthesized by conjugation of a synthetic collagen fragment to the collagen binding protein CNA35via a protease-cleavable linker. Cleavage of the linker by MMP1 releases the intramolecular inhibition of the collagen binding site and restores its collagen binding capacity.

DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control.

Introduction of a (Cys)4 metal binding site at the dimerization interface of two fluorescent protein domains yields a chelating FRET sensor protein that shows a 2500-fold selectivity for Cd2+ over Zn2+ by taking advantage of their different ionic radii.

A modular self-assembly strategy is presented that allows the non-covalent synthesis of multivalent protein dendrimers using the strong interaction between choline-functionalized dendrimers and the choline binding protein C-LytA. Choline dendrimers displaying fusion proteins of C-LytA and the collagen binding protein CNA35 represent attractive multivalent targeting ligands for collagen imaging.

We previously reported the development of high affinity Zn2+ FRET sensors based on the Zn2+-mediated interaction between the CXXC motifs present in the copper chaperone proteins ATOX1 and WD4. By systematically substituting several of these cysteines for methionines, we constructed sensor variants that retain a high affinity for Cu+, while effectively abolishing their ability to form stable tetrahedral Zn2+ complexes.

Detection of antibodies is essential for the diagnosis of many disease states, including infectious diseases, autoimmune diseases and allergies. Most current antibody detection assays involve multistep detection schemes in which molecular recognition and signal generation are separate processes. A well-known example is the enzyme-linked immunosorbent assay (ELISA), which combines high sensitivity and specificity with strong signal amplification. However, ELISA and other heterogeneous methods require multiple, time-consuming washing and incubation steps, which limits their applicability in point-of-care diagnostics and high-throughput applications. In recent years, several new antibody detection strategies have been developed in which antibody binding and signal generation are integrated within a single biomolecular reporter. These strategies aim to rival ELISA in terms of sensitivity and specificity, while decreasing the time and effort required to perform an assay. Here, we review recent developments in this field according to their mechanism of action and discuss their advantages and limitations.

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1:1 antibody–ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide–dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide–dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.