Functionally modified (Deoxy)Ribonucleotides : synthesis and study of physicochemical and biological properties
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Abstract
Understanding and controlling the role of RNA in processes of human health and disease, apart from basic cellular mechanisms, is of immense value. Many severe cases are still in need for optimized therapy strategies, a gap, which could be filled in the near future by promising therapeutic RNA applications. Especially the design of tailored siRNA and mRNA strategies towards personal therapy presents an encouraging point of vantage. The development of such techniques requires fundamental knowledge concerning physicochemical properties of RNA, as the implementation of molecular changes could influence the resulting biological activity in manifold directions.
The first project of the current thesis addressed this issue in assessing the influence of a terminal alkyne attached to the exocyclic amine of 2’-O-deoxycytidine phosphoramidites, which were applied in solid phase DNA synthesis of 22mers, exhibiting an analogous sequence to an eGFP-siRNA sense strand. The preferred orientation of this functional group and its influence on duplex stability were investigated via 2D-NMR spectroscopy and temperature dependent UV-absorption melting experiments, revealing significant changes of stability in the case of a double modified 5-methyl-2’-O-deoxycytidine. Additionally, the terminal alkyne served as a handle for the copper-I-catalyzed azide-alkyne cycloaddition (CuAAC) with a fluorescent dye.
Driven by the latest insights on the molecular mechanisms of TLR7 and 8 activation, which were suggested to be activated via two distinct triggers and pathways, the idea was born to address them simultaneously with one molecule combining both activating patterns, a small molecule TLR agonist and a single-stranded RNA, in one bidentate ligand to manipulate the innate immune system. To this end, the bioorthogonal CuAAC should present again the method of choice to link the desired molecules. Therefore, two small molecules of the imidazoquinoline family were prior equipped synthetically with azide-linkers for immobilization on designed functional alkyne-bearing mRNA molecules. The focus of this study was on the evaluation of cytokine release by human immune cells under the influence of the created new smTLRa-mRNA conjugates and their respective building blocks. Besides the described long and coding mRNAs, investigations involved also the modification and application of siRNA-conjugates, representing short non-coding RNAs. Bearing a potential therapeutic application in mind, which would involve the original function of the applied RNA species, as part of the translational machinery and RNAi mediated expression control, effects by the implemented RNA-modulations on these biological properties were investigated.