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Nucleolytic ribozymes utilize general acid‐base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2′‐OH group to attack the scissile phosphate (γ‐catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7‐fold decreased rate. Therefore, the common view that γ‐catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ‐catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines. [ABSTRACT FROM AUTHOR]

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis, tRNAAsn, tRNATyr, and tRNAAsp. It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl‐queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q‐family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q‐nucleoside family. [ABSTRACT FROM AUTHOR]

N6‐isopentenyladenosine (i6A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6A. [ABSTRACT FROM AUTHOR]

Abstract: Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC‐MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high‐resolution mass spectrometry. The structure of msms2i6A (2‐methylthiomethylenethio‐N6‐isopentenyl‐adenosine) was confirmed by methione‐D3 feeding experiments and by synthesis of the nucleobase. The msms2i6A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2i6A by the radical‐SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2i6A from i6A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry. [ABSTRACT FROM AUTHOR]

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3′ end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv. The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity. [ABSTRACT FROM AUTHOR]