Synthetic Genes For Dynamic Regulation Of DNA-Based Receptors.

We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA-based receptors: a triplex-forming DNA complex, an ATP-binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

Enzyme-Linked DNA Displacement (ELIDIS) Assay for Ultrasensitive Electrochemical Detection of Antibodies.

Here we report the development of a method for the electrochemical ultrasensitive detection of antibodies that couples the programmability and versatility of DNA-based systems with the sensitivity provided by enzymatic amplification. The platform, termed Enzyme-Linked DNA Displacement (ELIDIS), is based on the use of antigen-DNA conjugates that, upon the bivalent binding of a specific target antibody, induce the release of an enzyme-DNA hybrid strand from a preformed duplex. Such enzyme-DNA hybrid strand can then be electrochemically detected with a disposable electrode with high sensitivity. We applied ELIDIS to demonstrate the sensitive (limit of detection in the picomolar range), specific and multiplexed detection of five different antibodies including three clinically relevant ones. ELIDIS is also rapid (it only requires two reaction steps), works well in complex media (serum) and is cost-effective. A direct comparison with a commercial ELISA kit for the detection of Cetuximab demonstrates the promising features of ELIDIS as a point-of-care platform for antibodies detection.

Bispecific Antibody Detection Using Antigen-Conjugated Synthetic Nucleic Acid Strands.

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.

Synthetic Antigen-Conjugated DNA Systems for Antibody Detection and Characterization.

Antibodies are among the most relevant biomolecular targets for diagnostic and clinical applications. In this Perspective, we provide a critical overview of recent research efforts focused on the development and characterization of devices, switches, and reactions based on the use of synthetic antigen-conjugated DNA strands designed to be responsive to specific antibodies. These systems can find applications in sensing, drug-delivery, and antibody-antigen binding characterization. The examples described here demonstrate how the programmability and chemical versatility of synthetic nucleic acids can be used to create innovative analytical tools and target-responsive systems with promising potentials.

Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

Electrochemical Cell-Free Biosensors for Antibody Detection.

We report here the development of an electrochemical cell-free biosensor for antibody detection directly in complex sample matrices with high sensitivity and specificity that is particularly suitable for point-of-care applications. The approach is based on the use of programmable antigen-conjugated gene circuits that, upon recognition of a specific target antibody, trigger the cell-free transcription of an RNA sequence that can be consequently detected using a redox-modified probe strand immobilized to a disposable electrode. The platform couples the features of high sensitivity and specificity of cell-free systems and the strength of cost-effectiveness and possible miniaturization provided by the electrochemical detection. We demonstrate the sensitive, specific, selective, and multiplexed detection of three different antibodies, including the clinically-relevant Anti-HA antibody.

Programmable Cell-Free Transcriptional Switches for Antibody Detection.

We report here the development of a cell-free in vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA aptamer. The system couples the unique programmability and responsiveness of DNA-based systems with the specificity and sensitivity offered by in vitro genetic circuitries and commercially available transcription kits. We demonstrate that cell-free transcriptional switches can efficiently measure antibody levels directly in blood serum. Thanks to the programmable nature of the sensing platform, the method can be adapted to different antibodies: we demonstrate here the sensitive, rapid, and cost-effective detection of three different antibodies and the possible use of this approach for the simultaneous detection of two antibodies in the same solution.

Protein-Protein Communication Mediated by an Antibody-Responsive DNA Nanodevice.

We report here the rational design and optimization of an antibody-responsive, DNA-based device that enables communication between pairs of otherwise non-interacting proteins. The device is designed to recognize and bind a specific antibody and, in response, undergo a conformational change that leads to the release of a DNA strand, termed the "translator," that regulates the activity of a downstream target protein. As proof of principle, we demonstrate antibody-induced control of the proteins thrombin and Taq DNA polymerase. The resulting strategy is versatile and, in principle, can be easily adapted to control protein-protein communication in artificial regulatory networks.

Single antibody detection in a DNA origami nanoantenna.

DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm.

Programmable, Multiplexed DNA Circuits Supporting Clinically Relevant, Electrochemical Antibody Detection.

Current health emergencies have highlighted the need to have rapid, sensitive, and convenient platforms for the detection of specific antibodies. In response, we report here the design of an electrochemical DNA circuit that responds quantitatively to multiple specific antibodies. The approach employs synthetic antigen-conjugated nucleic acid strands that are rationally designed to induce a strand displacement reaction and release a redox reporter-modified strand upon the recognition of a specific target antibody. The approach is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies), and selective (the platform can be employed in complex media, including 90% serum). The programmable nature of the strand displacement circuit makes it also versatile, and we demonstrate here the detection of five different antibodies, including three of which are clinically relevant. Using different redox reporters, we also show that the antibody-responsive circuit can be multiplexed and responds to different antibodies in the same solution without crosstalk.

Rational Control of the Activity of a Cu2+-Dependent DNAzyme by Re-engineering Purely Entropic Intrinsically Disordered Domains.

The function and activity of many proteins is finely controlled by the modulation of the entropic contribution of intrinsically disordered domains that are not directly involved in any recognition event. Inspired by this mechanism, we demonstrate here that we could finely regulate the catalytic activity of a model DNAzyme (i.e., a synthetic DNA sequence with enzyme-like properties) by rationally introducing intrinsically disordered nucleic acid portions in its original sequence. More specifically, we have re-engineered here the well-characterized Cu2+-dependent DNAzyme that catalyzes a self-cleavage reaction by introducing a poly(T) linker domain in its sequence. The linker is not directly involved in the recognition event and connects the two domains that fold to form the catalytic core. We demonstrate that the enzyme-like activity of this re-engineered DNAzyme can be modulated in a predictable and fine way by changing the length, and thus entropy, of such a linker domain. Given these attributes, the rational design of intrinsically disordered domains could expand the available toolbox to achieve a control of the activity of DNAzymes and, in analogy, ribozymes through a purely entropic contribution.

Publisher Correction: Orthogonal regulation of DNA nanostructure self-assembly and disassembly using antibodies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Orthogonal regulation of DNA nanostructure self-assembly and disassembly using antibodies.

Here we report a rational strategy to orthogonally control assembly and disassembly of DNA-based nanostructures using specific IgG antibodies as molecular inputs. We first demonstrate that the binding of a specific antibody to a pair of antigen-conjugated split DNA input-strands induces their co-localization and reconstitution into a functional unit that is able to initiate a toehold strand displacement reaction. The effect is rapid and specific and can be extended to different antibodies with the expedient of changing the recognition elements attached to the two split DNA input-strands. Such an antibody-regulated DNA-based circuit has then been employed to control the assembly and disassembly of DNA tubular structures using specific antibodies as inputs. For example, we demonstrate that we can induce self-assembly and disassembly of two distinct DNA tubular structures by using DNA circuits controlled by two different IgG antibodies (anti-Dig and anti-DNP antibodies) in the same solution in an orthogonal way.

Programmable RNA-based systems for sensing and diagnostic applications.

The emerging field of RNA nanotechnology harnesses the versatility of RNA molecules to generate nature-inspired systems with programmable structure and functionality. Such methodology has therefore gained appeal in the fields of biosensing and diagnostics, where specific molecular recognition and advanced input/output processing are demanded. The use of RNA modules and components allows for achieving diversity in structure and function, for processing information with molecular precision, and for programming dynamic operations on the grounds of predictable non-covalent interactions. When RNA nanotechnology meets bioanalytical chemistry, sensing of target molecules can be performed by harnessing programmable interactions of RNA modules, advanced field-ready biosensors can be manufactured by interfacing RNA-based devices with supporting portable platforms, and RNA sensors can be engineered to be genetically encoded allowing for real-time imaging of biomolecules in living cells. In this article, we report recent advances in RNA-based sensing technologies and discuss current trends in RNA nanotechnology-enabled biomedical diagnostics. In particular, we describe programmable sensors that leverage modular designs comprising dynamic aptamer-based units, synthetic RNA nanodevices able to perform target-responsive regulation of gene expression, and paper-based sensors incorporating artificial RNA networks. Graphical Abstract ᅟ.

Allosteric DNA nanoswitches for controlled release of a molecular cargo triggered by biological inputs.

Here we demonstrate the rational design of a new class of DNA-based nanoswitches which are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nanoswitch that adopts two mutually exclusive conformations: a "Load" conformation containing a doxorubicin-intercalating domain and a "Release" conformation containing a duplex portion recognized by a specific transcription-factor (here Tata Binding Protein). The binding of the transcription factor pushes this conformational equilibrium towards the "Release" state thus leading to doxorubicin release from the nanoswitch. In our second model system we designed a similar stem-loop DNA-nanoswitch for which conformational change and subsequent doxorubicin release can be triggered by a specific antibody. Our approach augments the current tool kit of smart drug release mechanisms regulated by different biological inputs.

Antibody-powered nucleic acid release using a DNA-based nanomachine.

A wide range of molecular devices with nanoscale dimensions have been recently designed to perform a variety of functions in response to specific molecular inputs. Only limited examples, however, utilize antibodies as regulatory inputs. In response to this, here we report the rational design of a modular DNA-based nanomachine that can reversibly load and release a molecular cargo on binding to a specific antibody. We show here that, by using three different antigens (including one relevant to HIV), it is possible to design different DNA nanomachines regulated by their targeting antibody in a rapid, versatile and highly specific manner. The antibody-powered DNA nanomachines we have developed here may thus be useful in applications like controlled drug-release, point-of-care diagnostics and in vivo imaging.

Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm.

A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(ß-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.

Reversible Electrochemical Modulation of a Catalytic Nanosystem.

A catalytic system based on monolayer-functionalized gold nanoparticles (Au NPs) that can be electrochemically modulated and reversibly activated is reported. The catalytic activity relies on the presence of metal ions (Cd(2+) and Cu(2+) ), which can be complexed by the nanoparticle-bound monolayer. This activates the system towards the catalytic cleavage of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP), which can be monitored by UV/Vis spectroscopy. It is shown that Cu(2+) metal ions can be delivered to the system by applying an oxidative potential to an electrode on which Cu(0) was deposited. By exploiting the different affinity of Cd(2+) and Cu(2+) ions for the monolayer, it was also possible to upregulate the catalytic activity after releasing Cu(2+) from an electrode into a solution containing Cd(2+) . Finally, it is shown that the activity of this supramolecular nanosystem can be reversibly switched on or off by oxidizing/reducing Cu/Cu(2+) ions under controlled conditions.

Electronic control of DNA-based nanoswitches and nanodevices.

Here we demonstrate that we can rationally and finely control the functionality of different DNA-based nanodevices and nanoswitches using electronic inputs. To demonstrate the versatility of our approach we have used here three different model DNA-based nanoswitches triggered by heavy metals and specific DNA sequences and a copper-responsive DNAzyme. To achieve electronic-induced control of these DNA-based nanodevices we have applied different voltage potentials at the surface of an electrode chip. The applied potential promotes an electron-transfer reaction that releases from the electrode surface a molecular input that ultimately triggers the DNA-based nanodevice. The use of electronic inputs as a way to finely activate DNA-based nanodevices appears particularly promising to expand the available toolbox in the field of DNA nanotechnology and to achieve a better hierarchical control of these platforms.