Tobias Santner

2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling.

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

Synthesis of (6-13C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics

We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed.

Pseudoknot Preorganization of the PreQ1 Class I Riboswitch

To explore folding and ligand recognition of metabolite-responsive RNAs is of major importance to comprehend gene regulation by riboswitches. Here, we demonstrate, using NMR spectroscopy, that the free aptamer of a preQ(1) class I riboswitch preorganizes into a pseudoknot fold in a temperature- and Mg(2+)-dependent manner. The preformed pseudoknot represents a structure that is close to the ligand-bound state and that likely represents the conformation selected by the ligand. Importantly, a defined base pair mutation within the pseudoknot interaction stipulates whether, in the absence of ligand, dimer formation of the aptamer competes with intramolecular pseudoknot formation. This study pinpoints how RNA preorganization is a crucial determinant for the adaptive recognition process of RNA and ligand.

Efficient access to 3'-terminal azide-modified RNA for inverse click-labeling patterns.

Labeled RNA becomes increasingly important for molecular diagnostics and biophysical studies on RNA with its diverse interaction partners, which range from small metabolites to large macromolecular assemblies, such as the ribosome. Here, we introduce a fast synthesis path to 3′-terminal 2′-O-(2-azidoethyl) modified oligoribonucleotides for subsequent bioconjugation, as exemplified by fluorescent labeling via Click chemistry for an siRNA targeting the brain acid-soluble protein 1 gene (BASP1). Importantly, the functional group pattern is inverse to commonly encountered alkyne-functionalized “click”-able RNA and offers increased flexibility with respect to multiple and stepwise labeling of the same RNA molecule. Additionally, our route opens up a minimal step synthesis of 2′-O-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for N-hydroxysuccinimide (NHS) ester-based conjugations.

Enzymatic synthesis of 2'-methylseleno-modified RNA

Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 2′-methylseleno (2′-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 2′-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 2′-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 2′-methylseleno-2′-deoxyribonucleoside triphosphates of cytosine and uridine (2′-SeCH3–CTP and 2′-SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 2′-SeCH3–CMP and 2′-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 2′-SeCH3-modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.

New Insights into Gene Regulation—High‐Resolution Structures of Cobalamin Riboswitches

Since the discovery of the first mRNA riboswitches around 2002, the concept that small molecules, mostly metabolites, directly bind to the nascent RNA chain and thereby trigger mutually exclusive folding pathways has been confirmed for many bacteria as well as for other organisms. This interaction results in either upor down-regulation of the corresponding gene. The nature of ligands participating in such a strategy is diverse, ranging from enzyme cofactors and nucleotide precursors, to amino acids and ions. They include adenosylcobalamin (AdoCbl), thiamine pyrophosphate, adenine, guanine, 7-aminomethyl-7-deazaguanine (preQ1), Sadenosylmethionine, S-adenosylhomocysteine, adenosine triphosphate, lysine, glycine, glutamine, tetrahydrofolate, flavin mononucleotide, cyclic diguanylate, glucosamine-6-phosphate, fluoride and magnesium ions, and most likely others will emerge in the future. The sizes of metabolite-binding mRNA domains range from 34 nucleotides for preQ1 [3] to about 200 nucleotides for AdoCbl. Strikingly, for almost all above-mentioned riboswitches the three-dimensional structures of the aptamer domains bound to the dedicated ligands have been solved by X-ray crystallography at high-resolution and thus provide a platform to comprehend the molecular mechanism of these systems. However, the 3’-adjoining expression platforms often elude attempts of crystallization, as many ligand-free aptamers do. This reflects the intrinsic dynamic nature of free riboswitches and is also in line with the fact that the expression platforms are much less conserved in sequence than aptamer sequences are. The former have evolved to satisfy structural features that are required to act at different modes of gene expression, predominantly at transcriptional or translational levels, but also through splice-site control, mRNA decay, and likely other mechanisms yet to be discovered. In this context, it is understandable that the largest and most complex riboswitch known to date (which was actually discovered as one of the first representatives, if not the first one) has resisted three-dimensional resolution and elucidation of its molecular mechanism for a decade. Very recently, the research groups lead by R. Batey and A. Serganov independently obtained high-resolution X-ray structures of cobalamin riboswitches and, together with a functional study by T. Pan and co-workers earlier this year on the ligandinduced folding pathway, these investigations now provide detailed molecular insights into the response mechanism of mRNAs specific for cobalamins. A first important finding in this recent work is that AdoCbl is not the only B12 cofactor derivative [9] that binds to this riboswitch family with high affinity. In cell-based assays, Batey and co-workers discovered that several cobalamin riboswitches from marine cyanobacterial and environmental (env) genomes from ocean-surface samples exhibit much higher affinities to methylcobalamin (MeCbl) and aquocobalamin (AqCbl), and clearly discriminate against AdoCbl (Figure 1). This distinction, which is directly related to the

The synthesis of 2′-methylseleno adenosine and guanosine 5′-triphosphates

Graphical abstract