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Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2- 19 F, 2- 13 C]-adenosine, which allow the incorporation of aromatic 19 F- 13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2- 19 F, 2- 13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2- 19 F, 2- 13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2- 19 F, 2- 13 C]-A-labeled RNA featured sharper resonances than the [2- 1 H, 2- 13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2- 19 F, 2- 13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2- 19 F, 2- 13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fluorine labeling of ribonucleic acids (RNA) in conjunction with 19 F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF 3 guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF 3 modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF 3 label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF 3 modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF 3 modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF 3 nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF 3 group without any restrictions in placing the modification into the RNA target sequence., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.

We present the access to [5- 19 F, 5- 13 C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5- 19 F, 5- 13 C]-pyrimidine labels into five RNAs-the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic 19 F- 13 C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13 C- 19 F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5- 19 F, 5- 13 C]/[5- 19 F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19 F- 13 C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [ 19 F, 13 C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+., Competing Interests: H.M.A. is an advisor and holds an ownership interest in Nymirum Inc., an RNA- based drug discovery company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs. mRNA modifications within coding sequences can either directly or indirectly interfere with protein synthesis. In order to investigate the roles of various natural occurring modified nucleotides, we site-specifically introduced them into the coding sequence of reporter mRNAs and subsequently translated them in HEK293T cells. The analysis of the respective protein products revealed a strong position-dependent impact of RNA modifications on translation efficiency and accuracy. Whereas a single 5-methylcytosine (m⁵C) or pseudouridine () did not reduce product yields, N ¹-methyladenosine (m¹A) generally impeded the translation of the respective modified mRNA. An inhibitory effect of 2' O -methlyated nucleotides (Nm) and N ⁶-methyladenosine (m⁶A) was strongly dependent on their position within the codon. Finally, we could not attribute any miscoding potential to the set of mRNA modifications tested in HEK293T cells., Competing Interests: The authors declare no conflicts of interest.

Watson-Crick like G-U mismatches with tautomeric G enol or U enol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo 5 U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo 5 U enol -G mismatches. A detailed investigation on the influence of the cmo 5 U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo 5 U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo 5 U enol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo 5 U - -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

N 6 -Methyladenosine (m 6 A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. In addition to recruiting proteins, m 6 A can modulate RNA function by destabilizing base pairing. Here, we show that when neighbored by a 5' bulge, m 6 A stabilizes m 6 A-U base pairs, and global RNA structure by ~1 kcal mol -1 . The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3' neighbor through a conformation that is stabilized by Mg 2+ . A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m 6 A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m 6 A antibodies, both readily recognize m 6 A in this newly identified motif. The results uncover potentially abundant and functional m 6 A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data.