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Improving the catalytic repertoire of DNA enzymes

Applicant Hollenstein Marcel
Number 144595
Funding scheme Ambizione
Research institution Departement für Chemie und Biochemie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Organic Chemistry
Start/End 01.11.2012 - 31.10.2013
Approved amount 158'421.00
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Keywords (5)

unnatural nucleotides; in vitro selection; bioorganic chemistry; nucleic acids ; deoxyribozymes

Lay Summary (English)

Lead
Lay summary

Deoxyribonucleoside triphosphates (dNTPs) are activated building blocks that are fundamental in the polymerase-mediated natural biosynthesis of DNA. Chemical modification of triphosphates at various locations followed by polymerization is a convenient and versatile way for the introduction of functional groups into nucleic acids. In addition, if the modified dNTPs are substrates for polymerases in both primer extension reactions (PEX) and polymerase chain reactions (PCR), then they can serve in SELEX (Systematic Enrichment of Ligands by Exponential Enrichment) for the generation of oligonucleotides binding to specific targets (aptamers) or catalytic DNAs (DNAzymes). In SELEX and related methods of combinatorial methods of in vitro selection, a large population of oligonucleotides (10^12 sequences) is examined in parallel for binding affinity or catalytic properties.

The projected work is heavily based on the original grant (PZ00P2_126430) and involves the use of modified dNTPs in in vitro selection experiments for the discovery of catalytic nucleic acids. Indeed, I have synthesized a variety of dNTPs bearing residues that are of primordial importance in organocatalysis. In addition, all these dNTPs were shown to be good substrates for both PEX and PCR and can thus be used in SELEX for the generation of DNAzymes catalyzing organic reactions in water. Moreover, dNTPs bearing carboxylic acid, imidazole, and guanidine residues are currently being synthesized and could be used in the selection of DNAzymes capable of catalyzing the scission of amide bond linkages.

 

In summary, the ultimate goal of this SNF grant is the discovery of DNAzymes with hitherto unknown properties, which in turn 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, while DNAzymes catalyzing organocatalytic reactions in water could be converted into valuable synthetic tools.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues - synthesis and biochemical characterization
Hollenstein Marcel (2013), Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues - synthesis and biochemical characterization, in ORGANIC & BIOMOLECULAR CHEMISTRY, 11(31), 5162-5172.
Toward the Combinatorial Selection of Chemically Modified DNAzyme RNase A Mimics Active Against all-RNA Substrates
Hollenstein Marcel, Hipolito Christopher J., Lam Curtis H., Perrin David M. (2013), Toward the Combinatorial Selection of Chemically Modified DNAzyme RNase A Mimics Active Against all-RNA Substrates, in ACS COMBINATORIAL SCIENCE, 15(4), 174-182.
Polymerize it all!
Hollenstein Marcel, Polymerize it all!, in OA Biochemistry.

Collaboration

Group / person Country
Types of collaboration
University of British Columbia Canada (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Ambizione meeting Talk given at a conference 20.08.2013 Berne, Switzerland Hollenstein Marcel;


Associated projects

Number Title Start Funding scheme
126430 Improving the catalytic repertoire of DNA enzymes 01.11.2009 Ambizione
108568 En route vers l'élaboration de déoxyribozymes possédant la capacité de condenser par une ligation 3'-5' 01.05.2005 Fellowships for prospective researchers

Abstract

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 catalysts that may find application in organic synthesis. The projects disclosed in this proposal are essentially based on the results obtained in the original Ambizione grant and allow for an extension to new research avenues, in particular, the development of DNAzymes with hitherto unknown activities. Indeed, after mastering the rather challenging synthesis of complexly modified triphosphates, in vitro selection experiments will be carried out in the proposed continuation of the Ambizione grant, to seek for DNAzymes that are able to catalyze the scission of amide-bond linkages and the Henry reaction. These findings could have an important impact on the fields of catalytic nucleic acids, artificial peptidases, and organocatalysis.
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