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MADNA Report Summary

Project ID: 280010
Funded under: FP7-IDEAS-ERC
Country: Netherlands

Final Report Summary - MADNA (Modular Assembly of DNA-based systems; bio-inspired artificial allosteric assemblies)

Metabolic pathways are tightly regulated to control the concentration of metabolites in the cell. While a number of different methods are used to achieve such regulation, perhaps the most fascinating is control of enzyme activity through allostery, in which binding of an effector molecule causes a structural change of the protein resulting in either an increase (positive) or decrease (negative allostery) of the catalytic activity.
In the MADNA project the aim was the development of biomolecular artificial allosteric systems, principally using DNA as the scaffold to affect a predictable structural change in an enzyme or a (bio-)molecular system. During the course of the project, also metal ions were investigated as the effector.

The design of the systems involved incorporating a synthetic host into a biomolecule such as protein (enzyme) or pore forming peptides. A significant part of the project was devoted to developing the methodology to construct such as artificial allosteric systems. For example, novel DNA conjugation reactions were developed.

One of the most important achievements of MADNA was the development of several new approaches towards the design of artificial enzymes, which will also impact the field of bio-catalysis. The first method involved supramolecular assembly of artificial enzymes. In contrast to existing approaches, non-specific, moderate affinity binding metal complexes were used. This resulted in very efficient and selective enzymes. Moreover, it was shown that the dynamics and flexibility of the system is actually beneficial: the enzyme can “find” its most optimal conformation by itself.

Covalent methods for creating artificial enzymes were also developed. A breakthrough was the first examples of application of “expanded genetic code” methods to introduce unnatural metal binding or catalytically active residues in the proteins in vivo. In combination with novel computational design methods, this gave rise to several new artificial enzymes that were employed successfully in (enantioselective) catalysis of new-to-nature reactions.

These novel methods were combined for the construction of the first artificial allosteric metalloenzymes in which a regulatory domain was anchored covalently and the catalytic domain was introduced via supramolecular binding. This resulted in an enzyme that was inactive by itself, but was selectively activated by the presence of a specific metal ion, that is, iron(II), but not zinc(II).

Non-catalytic systems were developed using DNA as regulatory moiety. DNA G-quadruplex micelles were created and it was shown that, for certain designs, the quadruplexes could be disprupted by strand exchange with another DNA of complementary sequence. This resulted in disassembly of the micelles and release of a cargo, in this case a fluorescent dye, in response to a DNA sequence. The system is now being made responsive to small molecules by the use of structure switching DNA aptamers for the strand exchange.

DNA conjugates of a variant of the antimicrobial pore forming peptide alamethicin were developed. Upon combination of multiple alamethicin-DNA conjugates through duplex or quadruplex formation, preferential formation of certain pore sizes could be observed that also were longer lived than those of the peptide itself. Also these are being explored in strand exchange studies with aptamers.

Finally, novel chemical nucleases were developed that proved to be highly active against cancer cells. It was shown that the biological activity of these chemical nucleases was dependent on which metal was bound, the transport mechanism into the cell and the cellular localization. This has resulted in important knowledge for development of new metal based anti-cancer strategies. Efforts to control the activity of these chemical nucleases by light were inconclusive to date.

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