Project

bioorganic chemistry; DNA enzymes; Enzyme mimics; Modified Nucleobases

Lead
Lay summary
Since the identification of DNA as the carrier of genetic information and its key role in the central dogma of molecular biology, numerous applications of nucleic acids, often very different from these original properties, have emerged. It is in regards to the catalytic properties of DNA that this project is focused. Indeed, DNAzymes are molecules able to catalyze various reactions and unlike their RNA counterparts, do not have any precedent in nature. DNAzymes are usually obtained by molecular evolution-based selection techniques which basically aim at achieving Darwinian evolution on a large population of DNA molecules in a test tube. The goals of the projects are to use chemically modified nucleic acids in such selection techniques, in order to unravel DNAzymes that mimic the activity of certain protein enzymes. In particular, using DNA adorned with carboxylic residues could lead to a DNAzyme acting as an artificial aspartic protease, which is able to catalyze the cleavage of amide bonds. Moreover, the use of another modified nucleic acid analog (LNA), known to stabilize DNA duplexes, could lead to the identification of DNAzymes mimicking the enzyme RNase A, which cleaves single stranded RNA strands. This project could provide significant improvements in the field of catalytic nucleic acids and could have deep ramifications in fundamental research and therapeutical applications. For instance, DNAzymes acting as artificial proteases could be used in the development of new drugs especially towards amyloid diseases such as Alzheimer's disease, while a DNAzyme cleaving RNA linkages could potentially be used as an anti-mRNA agent. Finally, the project will require a myriad of various techniques ranging from synthetic organic chemistry to the use of molecular biology tools. The combination of these techniques opens up realms of possibilities that could not be reached with only one of these alone.

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Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

DNAzymes are DNA molecules able to catalyze a variety of reactions and unlike their RNA counterpart, have no precedent in nature. Consequently, DNAzymes are obtained artificially by molecular evolution-based combinatorial techniques such as SELEX (systematic evolution of ligands by exponential enrichment). The prevalent application to which many in vitro selection experiments still strive, is the use of DNAzymes as catalytic antisense agents for the specific cleavage of mRNA leading to gene silencing. Even though the knowledge gathered on deoxyribozymes advances at a quick pace and numerous applications are emerging, important gaps still need to be filled. Indeed, the use of DNAzymes adorned with chemical functionalities could drastically increase the number and nature of the catalyzed chemical reactions. Moreover, due to the ease of selection for catalytic activity and synthetic scale-up, DNAzymes represent a potential interesting class of catalyst that may find application in organic synthesis, provided their solubility in organic media is increased. Finally, in vitro selections are often lengthy procedures that lead to self-cleaving deoxyribozymes that need to be re-engineered to trans-cleaving species to achieve multiple turnover catalysis. Consequently, new methods that could reduce the length of the procedure and yield directly trans-acting deoxyribozymes could be of high benefit.The general aim of the project presented herein, is to increase the potency and usefulness of DNAzymes as catalysts by increasing their catalytic and chemical repertoire, their solubility in organic media and their resistance to nuclease degradation. To this end, the project is divided in three sections, each of which dealing with one of these facets of the mentioned projected improvements:1) In vitro selection of an LNAzyme acting as an RNase mimic. The aim of this part of the project is to use known locked nucleic acid (LNA) triphosphate units for an in vitro selection experiment to discover a DNAzyme with enhanced catalytic properties and stability to nuclease degradation. 2) Selection of a DNAzyme with peptidase-like activity. This section questions whether it is possible to find a DNAzyme catalyzing the hydrolysis of an amide bond. To this effect, a PNA/DNA chimeric substrate will be used in an in vitro selection experiment. In addition, a modified dNTP bearing a hydrophobic residue is hypothesized to facilitate the in vitro selection discovery of a DNAzyme able to function in organic medium.3) Selection using transient functional groups. A disulfide-bearing dNTP analogue will be used as anchor for the temporary attachment of various functional groups that could mediate the cleavage of a ribonucleotide linkage. This strategy used in the context of in vitro selections, could allow for the rapid discovery of DNAzymes catalyzing a broad variety of reactions.