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Enzyme-conjugated DNA nanoswitches for biosensing applications

Periodic Reporting for period 2 - ENZYME-SWITCHES (Enzyme-conjugated DNA nanoswitches for biosensing applications)

Reporting period: 2021-10-01 to 2022-09-30

The present fellowship proposed the development of a new generation of enzyme-based biosensors for diagnostic applications. I proposed to use enzymes conjugated with synthetic DNA responsive nanoswitches, with the main goal of combining the high specificity of enzymes and the high programmability and versatility of DNA nanodevices,
According to the general idea, the enzyme will recognize a specific diagnostic target and will catalyze the formation of a product. Then the DNA responsive nanoswitch will be able to recognize and detect the enzymatic product.
As we have previously reported, the direct conjugation among a single enzyme and a single DNA nanoswitch could create issues in obtaining good results. Thus, in order to improve the communication among enzymes and DNA modules, we decided to study the possibility of using a polymeric DNA-based structure as a scaffold to co-localize biological macromolecules (i.e. enzymes) with several DNA-responsive modules (i.e. DNA nanoswitches). More specifically, to do that we have employed the well-known DNA tubular structures, tile-based DNA structures obtained through the self-assembly of double-cross-over DNA tiles (DAE-E). As already demonstrated, it is also possible to re-engineer different tiles with multiple functional groups and the same sticky ends, so that they can co-assemble and form a unique DNA scaffold with multiple functionalities
Nowadays, biosensors have become important devices in diagnosing and monitoring health. Among them, enzyme-based biosensors are one of the most successful examples, but they are still limited to measure few clinical targets. So the development of new strategies to create versatile platforms able to detect a wide range of clinical targets is needed.
To achieve this challenging purpose, several objectives have been defined:

• Design and optimization of the several pH-dependent DNA-based nanodevices.
• Study and characterization of aptamer-based DNA nanodevices.
• Synthesis and optimization of the enzyme-conjugated DNA nanoswitches.
• Design and optimization of the DNA scaffold to co-localize the enzyme and the reported DNA-nanodevice.
• Development of novel strategies to dynamically introduce functional groups in DNA structures.
• Characterization of the strand displacement reaction, one of the most used reaction to control the assembly of DNA structures.
• Demonstration of the orthogonality and versatility of the proposed strategies.
• Demonstration of the versatility of the proposed approach employing other enzyme-based biosensors.
In this project we have worked on the synthesis and optimization of the enzyme-conjugated DNA sensor. According to the general idea, the addition of the enzymatic substrate can cause the production or consumption of protons, so that the induced pH-change can be detected by a DNA device.
In order to obtain a better communication between the enzymes and the DNA devices, we investigated the possibility of employing a polymeric DNA-based structure as a scaffold to co-localize the enzyme with the DNA-responsive module. To do that, we have used the well-known DNA nanotubes, a design reported by Winfree, Rothemund and Franco. It is also already demonstrated that the disassembly and re-assembly of such structures can be easily controlled with the addition of several regulator strands.
In this reporting period we have reached several results. We first focused the attention on the characterization of several DNA-based systems, like pH-dependent triplex structures or also aptamer-based nanodevices, that can be controlled through different inputs, like pH, light, enzymes or small molecules.
We then demonstrated the possibility to functionalize the above described tile-based DNA structures with multiple functional groups. For example, we have employed pH-dependent DNA nanoswitch to obtain scaffolds that can be visualized in a pH-dependent way. Then, we have also developed novel strategies that allow to dynamically exchange the decoration on the DNA structures without destroying the scaffold itself.
Moreover, to better control the communication between the functional groups of the different tiles, we also investigated novel strategies to finely control the assembly/disassembly process of such DNA-based structures or also to trigger the re-organization of such DNA structures, for example from homopolymers to random copolymers and block copolymers. Specifically, we demonstrated novel strategies to achieve this re-organization in a spontaneous way by employing enzymatic or chemical dissipative reactions.
Finally, more recently, we are investigating the possibility of employing such tile-based DNA structures as nanomaterials to generate a readout signal in a lateral flow assay (LFA).
During this period I also participated to several international conferences, and among them I received two invitations, one as a finalist for the “European Young Chemist Award”, for which I received the Silver Medal.
One of the major goal in biomedical applications is the development of biosensors for sensitive, specific, rapid and easy-to-use measurements of diagnostic biomarkers. Biosensors provide a key role in diagnosing and monitoring, and among them the enzyme-based biosensors are the most representative and successful examples. We proposed here to combine the positive features of this field with the high programmability and versatility of DNA as biomaterial to achieve innovative bio-sensing devices.
We started the characterization of a DNA-based structure that can be used as a scaffold to co-localize enzymes and responsive DNA nanodevices. These structures are DNA-based polymer systems consisting of multiple assembling monomers (DNA tiles) that can be organized with different distributions, forming homopolymers (by one type of tile) random copolymers (the two tiles are randomly distributed) and block copolymers (containing separated segments of the same tiles). In this period we also characterized several DNA-based nanodevices, that can also be used to functionalize such DNA structures. Moreover we demonstrated different strategies that allow to control the reorganization of tiles distribution, with both, enzymatic or chemical spontaneous mechanisms.
Given the good results obtained by employing polymeric-DNA structures as scaffold to co-localize enzymes and DNA responsive modules, together with the nice results achieved when DNA scaffolds are used as nanomaterials in lateral flow assays (LFAs), we think that the developed strategies could significantly help in reaching the main objectives of this proposal. In addition, the high versatility of the strategies that we have demonstrated here allow the rapid detection of a wide range of clinical targets, so all these features could significantly impact the development of bio-sensing devices, narrowing the gap between diagnosis, treatment and control of diseases.
Strategy for appending enzymes to DNA structures