Escherichia coli tRNA 2-Selenouridine Synthase (SelU): Elucidation of Substrate Specificity to Understand the Role of S-Geranyl-tRNA in the Conversion of 2-Thio- into 2-Selenouridines in Bacterial tRNA

The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions

Hydrophilic Polyhedral Oligomeric Silsesquioxane, POSS(OH)32, as a Complexing Nanocarrier for Doxorubicin and Daunorubicin

A novel strategy, recently developed by us, to use polyhedral oligomeric silsesquioxanes (POSS) as an anti-cancer drug carrier is presented. Anthracycline:POSS complexes were prepared by simple co-addition of doxorubicin (DOX) or daunorubicin (DAU) with hydrophilic POSS(OH)32. Co-delivery of POSS and anthracyclines led to higher anti-cancer activity towards HeLa (cervical cancer endothelial) and MCF-7 (human breast adenocarcinoma) cell lines. The obtained supramolecular hybrid complexes were characterised by nuclear magnetic resonance (NMR) spectroscopy (nuclear Overhauser effect spectroscopy [NOESY] and homonuclear correlation spectroscopy [COSY]), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). The two-dimensional (2D) NOESY spectra of the complexes showed the cross-correlation peaks for hydroxyl groups of POSS (~4.3–4.8 ppm) with OH groups of DOX and DAU. FTIR showed that hydroxyl group of POSS can interact with amine and hydroxyl groups of DOX and DAU. The viability of HeLa and MCF-7 was analysed with the MTT assay to evaluate the cytotoxicity of free DOX and DAU and the relevant complexes with POSS at different molar ratios. At a low DOX concentration (2.5 µM), for molar ratios 1:1, 1:4, and 1:8 (POSS:DOX), the complexes showed two and three times higher cytotoxicity towards HeLa and MCF-7 cells, respectively, than DOX itself after both 24- and 48-h incubation. The 1 µM concentration for a 1:4 POSS:DOX molecular ratio and the 2.5 µM concentration for all complexes were more toxic towards MCF-7 cells than free DOX after 48-h incubation. In the case of POSS:DAU complexes, there was higher toxicity than that of free drug after 48-h incubation. It can be concluded that the formation of non-covalent complexes increases toxicity of anthracycline drugs towards Hela and MCF-7 cells. The novel complexes are inexpensive to prepare and more effective than free drugs at low systemic toxicity