Machine learning model for sequence-driven DNA G-quadruplex formation.

We describe a sequence-based computational model to predict DNA G-quadruplex (G4) formation. The model was developed using large-scale machine learning from an extensive experimental G4-formation dataset, recently obtained for the human genome via G4-seq methodology. Our model differentiates many widely accepted putative quadruplex sequences that do not actually form stable genomic G4 structures, correctly assessing the G4 folding potential of over 700,000 such sequences in the human genome. Moreover, our approach reveals the relative importance of sequence-based features coming from both within the G4 motifs and their flanking regions. The developed model can be applied to any DNA sequence or genome to characterise sequence-driven intramolecular G4 formation propensities

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

Screening mutant libraries of T7 RNA polymerase for candidates with increased acceptance of 2'-modified nucleotides

We present a screening assay based on fluorescence readout for the directed evolution of T7 RNA polymerase variants with acceptance of 2'-modified nucleotides. By using this screening we were able to identify a T7 RNA polymerase mutant with increased acceptance of 2'-methylseleno-2'-deoxyuridine 5'-triphosphate

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′-<i>O</i>-(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 (<i>BASP1</i>). 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′-<i>O</i>-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for <i>N</i>-hydroxysuccinimide (NHS) ester-based conjugations

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′-<i>endo</i> 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′-<i>O</i>-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

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

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2′-SeCH3-uridine and 2′-SeCH3-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2′-methylseleno-2′-deoxyadenosine and -guanosine 5′-triphosphates (2′-SeCH3-ATP and 2′-SeCH3-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases