Wouter Engelen

eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn2+ Imaging

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.

DNA-Directed Control of Enzyme–Inhibitor Complex Formation: A Modular Approach to Reversibly Switch Enzyme Activity

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.

Antibody-controlled actuation of DNA-based molecular circuits

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Controlling protein activity by dynamic recruitment on a supramolecular polymer platform

Nature uses dynamic molecular platforms for the recruitment of weakly associating proteins into higher-order assemblies to achieve spatiotemporal control of signal transduction. Nanostructures that emulate this dynamic behavior require features such as plasticity, specificity and reversibility. Here we introduce a synthetic protein recruitment platform that combines the dynamics of supramolecular polymers with the programmability offered by DNA-mediated protein recruitment. Assembly of benzene-1,3,5-tricarboxamide (BTA) derivatives functionalized with a 10-nucleotide receptor strand into µm-long supramolecular BTA polymers is remarkably robust, even with high contents of DNA-functionalized BTA monomers and associated proteins. Specific recruitment of DNA-conjugated proteins on the supramolecular polymer results in a 1000-fold increase in protein complex formation, while at the same time enabling their rapid exchange along the BTA polymer. Our results establish supramolecular BTA polymers as a generic protein recruitment platform and demonstrate how assembly of protein complexes along the supramolecular polymer allows efficient and dynamic control of protein activity.DNA-origami allows the precise recruitment of DNA-protein conjugates but lacks the dynamics found in natural protein assemblies. Here the authors present a synthetic polymer platform that combines the dynamics of supramolecular polymers with the programmability of DNA-mediated protein recruitment.

Hierarchical control of enzymatic actuators using DNA-based switchable memories

Inspired by signaling networks in living cells, DNA-based programming aims for the engineering of biochemical networks capable of advanced regulatory and computational functions under controlled cell-free conditions. While regulatory circuits in cells control downstream processes through hierarchical layers of signal processing, coupling of enzymatically driven DNA-based networks to downstream processes has rarely been reported. Here, we expand the scope of molecular programming by engineering hierarchical control of enzymatic actuators using feedback-controlled DNA-circuits capable of advanced regulatory dynamics. We developed a translator module that converts signaling molecules from the upstream network to unique DNA strands driving downstream actuators with minimal retroactivity and support these findings with a detailed computational analysis. We show our modular approach by coupling of a previously engineered switchable memories circuit to downstream actuators based on β-lactamase and luciferase. To the best of our knowledge, our work demonstrates one of the most advanced DNA-based circuits regarding complexity and versatility.Naturally evolved regulatory circuits have hierarchical layers of signal generation and processing. Here, the authors emulate these networks using feedback-controlled DNA circuits that convert upstream signaling to downstream enzyme activity in a switchable memories circuit.

DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formation

Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.

Accelerating DNA-Based Computing on a Supramolecular Polymer

Dynamic DNA-based circuits represent versatile systems to perform complex computing operations at the molecular level. However, the majority of DNA circuits relies on freely diffusing reactants, which slows down their rate of operation substantially. Here we introduce the use of DNA-functionalized benzene-1,3,5-tricarboxamide (BTA) supramolecular polymers as dynamic scaffolds to template DNA-based molecular computing. By selectively recruiting DNA circuit components to a supramolecular BTA polymer functionalized with 10-nucleotide handle strands, the kinetics of strand displacement and strand exchange reactions were accelerated 100-fold. In addition, strand exchange reactions were also favored thermodynamically by bivalent interactions between the reaction product and the supramolecular polymer. The noncovalent assembly of the supramolecular polymers enabled straightforward optimization of the polymer composition to best suit various applications. The ability of supramolecular BTA polymers to increase the efficiency of DNA-based computing was demonstrated for three well-known and practically important DNA-computing operations: multi-input AND gates, Catalytic Hairpin Assembly and Hybridization Chain Reactions. This work thus establishes supramolecular BTA polymers as an efficient platform for DNA-based molecular operations, paving the way for the construction of autonomous bionanomolecular systems that confine and combine molecular sensing, computation, and actuation.

An Ultracold Electron Source for Ultrafast Electron Diffraction Experiments

We create ultrashort, ultracold electron bunches by accelerating electrons which are created by near-threshold photoionization of a cloud of laser-cooled atoms. With these bunches we can perform diffraction experiments of crystals of macromolecules.