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.

Response surface methodology (RSM) is a widely used mathematical and statistical technique for modeling and optimizing the process for the extraction of bioactive compounds. This review explains the optimization approach through the use of experimental design and empirical models for response prediction and the utilization of the desirability function for multiple response optimization. This paper also reviews recent studies on the application of RSM to optimize bioactive compound extraction processes such as conventional solvent extraction, microwave‐assisted extraction, supercritical fluid extraction, and ultrasound‐assisted extraction. Finally, the challenges associated with the use of RSM and the efforts made to improve RSM in the extraction process are also highlighted. Overall, this review informs many aspects of RSM that are occasionally ignored or insufficiently discussed with regard to the optimization of bioactive compound extraction processes, and it summarizes significant applications where RSM proved suitable. © 2022 Society of Chemical Industry.

Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA(Lys) is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m(2)G10 alone does not facilitate the folding of tRNA(Lys), but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m(1)A9 exceeds the contribution of m(1)A alone. This constitutes an unprecedented cooperative effect of two nucleotide modifications in the context of a naturally occurring RNA, which may be of general importance for tRNA structure and help understanding several recently described decay pathways for hypomodified tRNAs.