Studies on the Chemical Biology of Natural and Chemical Ribonucleotide Modifications

This work is centered on the synthesis and chemical recognition of modified RNA. Phosphoramidites of m5U, Ψ, m1G, m2G and m22G were synthesized and were incorporated into RNA oligomers by standard SPS. The synthesized oligomers were used for two purposes: (i) the total construction of tRNA via enzyme catalyzed ligation, and (ii) investigations of modifications in siRNAs. The construction of human mitochondrial tRNAIle (i) with and without modification was achieved in a one-pot 3 fragments ligation using T4-DNA-ligase with a DNA-splint. The yield for the ligation was 20-30 % and the products were further investigated for the stability of the tertiary structure via UV-melting curve analysis. The unmodified tRNA was found to be significantly less stable than the fully modified tRNA. In the second case (ii) RNAi efficiency and the immunostimulation in cells were investigated with 22 nucleotide long double-stranded siRNAs that were synthesized containing the modifications m1G, m2G, m22G, rT and in varying stochiometry. The investigations showed differential effects in knock-down as well as immunostimulation. In a related perspective, compounds for the chemical recognition of modifications in RNA were synthesized. Since the recognition of pseudouridine by 4-bromomethyl-coumarin derivates is of particular interest, 7-azido-4-bromomethyl-coumarin was obtained with an overall yield of 44 % in a 5-step synthesis. Another derivative, the biotinylated 4-bromomethyl-coumarin spaced with jeffamine-148 was obtained in a 6-step synthesis with an overall yield of 17 %, to allow the use of strepavidin for affinity separation. In the course of further investigations of coumarine conjugates, mild and bio-compatible conditions for the syntheses of rhodols and rhodamines were discovered. The conversion of fluorescein into the 3’-mesylated or 3’-6’-dimesylated derivate and the subsequent displacement of the mesylate with a nitrogenous nucleophile gave rise to rhodols or rhodamines, respectively. As these reaction conditions are compatible with biomolecular labeling, the tagging of polypeptides was investigated. Tri-and pentapeptides were synthesized, and dye-labeled via 3’/6’-xanthene substitution, and the product identity confirmed by MALDI-TOF-MS. Further syntheses via 3’/6’-xanthene substitution lead to moderate yields of 3',6'-bis(dimethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one, 3',6'-di(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one and the 3',6'-bis(2-(2-(2-aminoethoxy)ethoxy)ethyl amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light

DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position

International audienceThe carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct modification of the 5-position by electrophiles or by further derivatization of the 5-methylpyrimidines. Here, we review the structural diversity of these modified bases, the variety of cofactors that serve as carbon donors, and the common principles shared by enzymatic mechanisms generating them