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Enzyme-conjugated DNA nanoswitches for biosensing applications

Periodic Reporting for period 1 - ENZYME-SWITCHES (Enzyme-conjugated DNA nanoswitches for biosensing applications)

Reporting period: 2020-10-01 to 2021-09-30

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.
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. An important step forward in the field is the possibility to develop new versatile strategies and versatile platforms able to detect a wide range of clinical targets.
To achieve this challenging purpose, several objectives have been defined:

• Design and optimization of the DNA-based nanodevice.

• Synthesis and optimization of the enzyme-conjugated DNA nanoswitch.

• Design and optimization of the DNA scaffold to co-localize the enzyme and the reported DNA-nanodevice.

• Demonstration of the approach versatility with other enzyme-based biosensors. Other enzymes and other.
During this reporting period, we have worked on the synthesis and optimization of the enzyme-conjugated DNA nanoswitch (Urease with pH-dependent nanoswitch). According to the general idea, the addition of the enzymatic substrate (urea) should cause the consume of protons that can be detected by the DNA device.
This system does not give good results, so we decided to engineer and characterize a DNA-based structure that can act as a scaffold able to co-localize several enzymatic macromolecules with several DNA nanoswitches. To do this, we 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 focused our attention in demonstrating a new strategy to finely control the re-organization of such DNA-based structures, between homopolymers, random copolymers and block copolymers. In a more recent work, we also demonstrated a strategy to achieve this re-organization is a spontaneous way.
Considering the possibility to decorate the DNA tiles with different functional ligands, we anticipate that this strategy will be useful for the development of multifunctional systems in which the target detection is determined by the relative organization of the functional groups in the structure. To demonstrate this, we started by re-organizing new functionalized DNA structures.
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 demonstrated a strategy to control and re-organize such tile distribution, also with an enzymatic-driven spontaneous mechanism.
Given the nice results obtained until now with such new DNA-based structures, we think that this strategy could significantly help in reaching the main objectives of this proposal, and impact the development of bio-sensing devices, narrowing the gap between diagnosis, treatment and control of diseases.
Strategy for appending enzymes to DNA structures