Chemical Synthesis of Site‐Specifically 2′‐Azido‐Modified RNA and Potential Applications for Bioconjugation and RNA Interference

Aigner, Michaela; Hartl, Markus; Fauster, Katja; Steger, Jessica; Bister, Klaus; Micura, Ronald

doi:10.1002/cbic.201000646

Cited by 70 publications

(81 citation statements)

References 141 publications

(140 reference statements)

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“…13 Here, we expand these investigations toward 2′-azido cytidine and 2′-azido guanosine to further evaluate 2′-azido-modified oligoribonucleotides for siRNA applications. For reasons of comparability, we employed the same model system used previously to knock down the brain acid soluble protein 1 (BASP1) encoding gene by transient siRNA nucleofection in the chicken DF-1 cell line.…”

Section: Resultsmentioning

confidence: 99%

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Fauster¹,

Hartl²,

Santner³

et al. 2012

ACS Chem. Biol.

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106

116

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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.

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Section: Resultsmentioning

confidence: 99%

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Fauster¹,

Hartl²,

Santner³

et al. 2012

ACS Chem. Biol.

Self Cite

106

116

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“…3, 28, 29, 42, 46 With these synthetic approaches, N 3 and CN probes can be placed virtually anywhere providing a useful monitor of site-specific dynamics in environments such as the major groove, minor groove, and phosphate-sugar region. Herein, we demonstrate the effectiveness of a pair of vibrational reporters as tools to investigate structural and dynamic aspects of different regions of DNA and RNA aptamers using 2D IR.…”

Section: Introductionmentioning

confidence: 99%

Two-Dimensional Infrared Study of Vibrational Coupling between Azide and Nitrile Reporters in a RNA Nucleoside

et al. 2016

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The vibrations in the azide, N3, asymmetric stretching region and nitrile, CN, symmetric stretching region of 2’-azido-5-cyano-2’-deoxyuridine (N3CNdU) are examined by two-dimensional infrared spectroscopy (2D IR). At earlier waiting times, the 2D IR spectrum shows the presence of both vibrational transitions along the diagonal and off-diagonal cross peaks indicating vibrational coupling. The coupling strength was determined from the off-diagonal anharmoncity to be 66 cm−1 for the molecular distance of ~7.9 Å based on a structural map generated for this model system. In addition, the frequency-frequency correlation decay is detected, monitoring the solvent dynamics around each individual probe position. Overall, these vibrational reporters can be utilized in tandem to simultaneously track global structural information and fast structural fluctuations.

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“…The crude product was purified by column chromatography on silica gel, eluting with a 10-25% gradient of ethylacetate in pentane to provide 4.22 g (77%) of 32. (33) A solution of bromine (1.37 mL, 26.7 mmol) in dichloromethane (25 mL) was added dropwise to an ice-cold solution of 32 (4.22 g, 25.4 mmol) in dichloromethane (100 mL). Subsequently, 10% aqueous Na 2 S 2 O 3 (50 mL) is added.…”

Section: Endo-bicyclo[610]non-4-yn-9-ylmethyl (2-(2-hydroxyethoxy)ementioning

confidence: 99%

“…Most recently, Brown et al [25,26] further extended the latter approach by ON incorporation of aminoalkyl thymidine derivatives, followed by selective N-acylation with azide or cyclooctyne after cleavage from support. Alternative approaches for the preparation of azide-containing nucleotides-compromised by the incompatibility of azide with phosphoramidite chemistry-involve post-synthetic nucleophilic substitution [27][28][29] or selective diazotransfer reaction [30] or phosphonate-based coupling chemistry [31][32][33][34] However, a simple and general strategy for the on-support, automated synthesis of oligonucleotides with readily accessible building blocks, and suitable for introduction of any functional group (including cyclooctyne and azide), is still desirable.…”

Section: Introductionmentioning

confidence: 99%

Oligonucleotide Tagging for Copper-Free Click Conjugation

Jawalekar

Malik

Verkade³

et al. 2013

Molecules

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Abstract:Copper-free click chemistry between cyclooctynes and azide is a mild, fast and selective technology for conjugation of oligonucleotides. However, technology for site-specific introduction of the requisite probes by automated protocols is scarce, while the reported cyclooctynes are large and hydrophobic. In this work, it is demonstrated that the introduction of bicyclo[6.1.0]nonyne (BCN) into synthetic oligonucleotides is feasible by standard solid-phase phosphoramidite chemistry. A range of phosphoramidite building blocks is presented for incoporation of BCN or azide, either on-support or in solution. The usefulness of the approach is demonstrated by the straightforward and high-yielding conjugation of the resulting oligonucleotides, including biotinylation, fluorescent labeling, dimerization and attachment to polymer.

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Chemical Synthesis of Site‐Specifically 2′‐Azido‐Modified RNA and Potential Applications for Bioconjugation and RNA Interference

Cited by 70 publications

References 141 publications

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Two-Dimensional Infrared Study of Vibrational Coupling between Azide and Nitrile Reporters in a RNA Nucleoside

Oligonucleotide Tagging for Copper-Free Click Conjugation

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