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978-3-8439-2523-5, Reihe Organische Chemie
Studien zu synthetischen DNA Origami-Strukturen
148 Seiten, Dissertation Universität Stuttgart (2016), Softcover, A5
Nucleic acids allow for the generation of nanoscale objects by molecular self-assembly. The DNA origami method uses the single-stranded genome of the bacteriophage-derived vector M13 as scaffold that is 7249 nucleotides in length. This scaffold strand is typically folded by hybridizing it with more than 200 staple strands to give the final nanostructure. This method generates three-dimensional structures over 100 nm in length. However, the thermal und chemical lability hinders the use of DNA nanostructures in technical applications. This thesis focuses on the assembly of small origami structures, which are more thermostable than known DNA origami due to chemical modifications. The aim was to develop covalent cross-linking methods for increasing the stability of origami structures. Oligonucleotide synthesis on solid support is limited to DNA strands with lengths of up to 100-200 nucleotides. In this study, solid-phase synthesis was combined with chemical ligation to produce oligonucleotides with up to 600 nucleotides in length.
The first part of this thesis focused on the preparation of a small scaffold for DNA origami. The scaffold was generated by excising a segment from the vector M13mp18 with the help of restriction enzymes. The desired linear fragment, 704 nucleotides in length, was isolated in 60 % yield using two duplex-forming oligonucleotides and the enzymes EcoRI and BglII. Folding of this scaffold with 24 staple strands to the designed "4F-origami", which resembles the fingers of a human hand, was induced in high yields and verified by transmission electron microscopy.
To access chemically modified scaffolds for DNA origami, a second project focused on the synthesis of single-stranded DNA with several hundred nucleotides in length. A procedure based on assembly-directed chemical ligation was developed. Using an optimized version of a phosphoramidate ligation, the synthesis of a 600mer product was achieved by simultaneous ligation of six oligonucleotides in an assembled origami structure. Further, a so-called "CLICK"-ligation was tested. The copper-catalyzed cycloaddition reaction between a 5'-azidoterminal oligonucleotide with the 3'-propargyl group of a second oligonucleotide proceeded well in the spatially restricted ligation positions of a DNA origami structure. .