Universität zu Köln

Dörrenhaus, Robert ORCID: 0009-0006-4685-9939 (2025). From Molecular Design to Supramolecular Recognition: Development of C-Glycosides, a DNA-Based Biosensor and Studies on Halogen Bonding in DNA. PhD thesis, Universität zu Köln.

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Abstract

This thesis combines the progression efforts from molecular design to supramolecular recognition in DNA and RNA with different applications, presenting three projects, that collectively expand the toolkit for advanced biomolecular functionalization. Specifically, it describes the development of novel C-glycosidic artificial nucleic acids, the creation of an innovative DNA-based biosensor for short oligonucleotide detection using whispering gallery mode microlasers, and the investigation of halogen bonding interactions in DNA. Artificial base pairs have grown huge interest since its first mention in the 1960s and since the discovery of the first unnatural base pair in the 1990s to expand the canonical genetic alphabet. By introducing new artificial base pairs into the genetic code, ground-breaking efforts were made to enlarge the toolbox for structural investigation in RNA by site-specific labelling, for the development of new biopolymers, by broadening the informational storage capacity of DNA and even facilitating the creation of a semi-synthetic organism. However, some challenges for existing artificial base pairs still remain, such as the structural resembling in a natural Watson-Crick like geometry, which requires plane interaction forces between the nucleobases and also a higher stability of the artificial nucleosides under chemical conditions in synthetic DNA and RNA production and higher yielding synthetic methods. This work describes the development of the novel hydrophobic nucleoside CTPT3, which is successfully incorporated into DNA and RNA as an unnatural CTPT3 – NaM base pair. This artificial nucleoside features a hydrolysis stable C-glycosidic bond and was investigated on its stability compared to its paragon, the N-glycosidic TPT3. Furthermore, synthetic strategies for C-glycosidic bond formation have been systematically explored, with cross-coupling mechanisms elucidated to facilitate future integration of diverse C-glycosides into the expanded genetic alphabet. The yield for rNaM was enhanced by adapting these routes into its synthesis. In the second part, dIIPO was developed, which features a halogen bonding donor-acceptor moiety and was successfully incorporated into DNA as IIPO – oIPP base pair. Efforts on a high yielding route towards the nucleoside are presented, as well as in deep studies on an optimized method for primer extension, which facilitates a selective full-length DNA synthesis with incorporation of dIIPO TP opposite of doIPP. This breakthrough base pair not only expands the genetic alphabet, but also features halogen bonding attraction forces, which have never been described in enzymatic incorporation of artificial base pairs before. Moreover, it facilitates the possible emergence of more natural Watson-Crick-like geometries in comparison to earlier unnatural base pairs. In addition to structural investigations with artificial base pairs, investigations on the conformational processes of DNA in general are from interest, as well as the detection of short oligomers. This detection i.e. of diagnostic markers can be an important tool for early detection and prevention. For application, a sensitive method for DNA sensing in body liquids or even in cells would be beneficial. Furthermore, recent methods often rely on fluorophores, which are liable to quenching and are limited in the amount of different dyes. Within this work, a new concept for DNA sensing based on refractive index changes of whispering gallery mode microlasers was developed and its successful usage in solution was demonstrated. For this, a method for DNA functionalization of microlasers was developed and different setups tested. For structural investigations and possible future application in drug release systems, a hairpin substitution mechanism was studied and established. This work provides a compelling demonstration of the usage of whispering gallery mode microlasers for DNA sensing and structural investigation by a stable easy applicable DNA functionalization method and paves the way for more complex multiplex sensing by first functionalization efforts on nanodisks, a disc-shaped sub-μm laser, as sensors.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Dörrenhaus, Robert robert.doerrenhaus@googlemail.com https://orcid.org/0009-0006-4685-9939 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-804065
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Organic Chemistry
Subjects:	Chemistry and allied sciences
Uncontrolled Keywords:	Keywords Language DNA English Biosensor English Halogen Bonding English C-Glycosides English Nucleic Acids English Whispering Gallery Mode Microlaser English RNA English
Date of oral exam:	31 October 2025
Referee:	Name Academic Title Kath-Schorr, Stephanie Prof. Dr. Giernoth, Ralf Prof. Dr. Voskuhl, Jens Prof. Dr. Riemer, Jan Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/80406