Triple-Stranded DNA As a Structural Element in DNA Origami.

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex-triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex-triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex-triplex crossovers. We find that duplex-triplex crossovers can complement and fully replace the canonical duplex-duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.

A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

Antigen-Triggered Logic-Gating of DNA Nanodevices.

Synthetic nanoscale devices that reconfigure dynamically in response to physiological stimuli could offer new avenues for diagnostics and therapy. Here, we report a strategy for controlling the state of DNA nanodevices based on sensing antigens with IgG antibodies. To this end, we use IgG antibodies as structural elements to kinetically trap reconfigurable DNA origami structures in metastable states. Addition of soluble antigens displace the IgGs from the objects and triggers reconfiguration. We demonstrate this mechanism by antigen-triggered disassembly of DNA origami shells for two different IgGs and their cognate antigens, and we determined the corresponding dose response curves. We also describe the logic-gated actuation of DNA objects with combinations of antigens, as demonstrated with AND-type shells that disassemble only when two different antigens are detected simultaneously. We apply our system for the antigen-triggered release of molecular payload as exemplified by the release of virus particles that we loaded into the DNA origami shells. We expect our approach to be applicable in many types of DNA nanostructures and with many other IgG-antigen combinations.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

Advancing Biophysics Using DNA Origami.

DNA origami enables the bottom-up construction of chemically addressable, nanoscale objects with user-defined shapes and tailored functionalities. As such, not only can DNA origami objects be used to improve existing experimental methods in biophysics, but they also open up completely new avenues of exploration. In this review, we discuss basic biophysical concepts that are relevant for prospective DNA origami users. We summarize biochemical strategies for interfacing DNA origami with biomolecules of interest. We describe various applications of DNA origami, emphasizing the added value or new biophysical insights that can be generated: rulers and positioning devices, force measurement and force application devices, alignment supports for structural analysis for biomolecules in cryogenic electron microscopy and nuclear magnetic resonance, probes for manipulating and interacting with lipid membranes, and programmable nanopores. We conclude with some thoughts on so-far little explored opportunities for using DNA origami in more complex environments such as the cell or even organisms.

Programmable Bivalent Peptide-DNA Locks for pH-Based Control of Antibody Activity.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide-DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide-DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide-DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic 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.

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.

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 ß-lactamase and luciferase. To the best of our knowledge, our work demonstrates one of the most advanced DNA-based circuits regarding complexity and versatility.

Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy.

A photocrosslinkable protein G variant was used as an adapter protein to covalently and site-specifically conjugate an antibody and an Fc-fusion protein to an oligonucleotide. This modular approach enables straightforward decoration of DNA nanostructures with complex native proteins while retaining their innate binding affinity, allowing precise control over the nanoscale spatial organization of such proteins for in vitro and in vivo biomedical applications.

DNA-Specific Biosensors Based on Intramolecular ß-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-ß-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.

Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids.

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.

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.

eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging.

Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) 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 Zn(2+), but distinctly different concentrations have been reported when using these sensors to measure Zn(2+) 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 Zn(2+) 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 Zn(2+) in the cytosol but was also successfully used for measuring Zn(2+) 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 Zn(2+) 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-ß-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.