Studies on the hydrolytic stability of 2′-fluoroarabinonucleic acid (2′F-ANA)

Watts, Jonathan K.; Katolik, Adam; Viladoms, Júlia; Damha, Masad J.

doi:10.1039/b900443b

Cited by 58 publications

(51 citation statements)

References 48 publications

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“…4´-Fluoro nucleosides are relatively unexplored in the literature, investigated in only a handful of studies (36)(37)(38)(39)(40), most of which encountered acid instability of the glycosidic linkage, which precluded these molecules from further studies and incorporation within oligonucleotides. We showed that the 2´,4´-difluoro ribo (34) and arabino (35) nucleosides had enhanced acid stability owing to the presence of the 2´-fluorine (41). Furthermore, we showed that the electronegative 4´ fluorine reinforced the RNA-like (3´-endo) conformation of 2´-F-ribonucleosides through an additional anomeric effect (F4´-C4´-O4´).…”

Section: Introductionmentioning

confidence: 89%

Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

et al. 2015

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2′-Deoxy-2′,4′-difluorouridine (2′,4′-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (T m) data of duplexes was consistent with the 2′,4′-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on T m is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the “neutral” effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2′,4′-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2′,4′-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2′-F-ANA modifications, and as active as the 2′-F-RNA modified siRNAs.

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Section: Introductionmentioning

confidence: 89%

Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

et al. 2015

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“…Nevertheless, while the gymnotic activity of the LNA gapmers is high and extremely robust in tissue culture, it is uncertain whether these molecules are uniquely competent. Damha and co-workers 10,11,12,13,14,15 have synthesized antisense oligos that substitute 2′-deoxy, 2′-fluoro-β- D -arabinonucleic acid (2′F-ANA: arabinose is an epimer of ribose at C2′) for deoxyribose in the oligo chain. Because the 2′-fluorine atom does not significantly perturb duplex helix structure, phosphorothioate 2′F-ANA/RNA duplexes activate RNase H. 12,13 Furthermore, this ability may even be greater for 2′F-ANA/DNA phosphorothioate chimeras than for the corresponding deoxyribose phosphorothioate oligos.…”

Section: Introductionmentioning

confidence: 99%

Antisense 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (2′F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids

Souleimanian

Deleavey

Soifer

et al. 2012

Molecular Therapy - Nucleic Acids

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Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2′-deoxy-2′fluoroarabino nucleic acid (2′F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2′F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2′F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded oligos in vitro.

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“…The masses determined for peaks 4 through 7, which are poorly resolved, and 9 and 10, which are poorly resolved from the sense strand peak, correspond to the mass of the sense strand that has undergone loss of one adenine (observed: 117.1; calculated: 117.1) or one guanine (observed: 133.4; calculated: 133.1). All of these degradants can be formed through a mechanism involving an oxocarbonium ion intermediate (Watts et al, 2009) and this reaction generally leads to strand scission. Figure 11 (adapted from Watts and Burrows) illustrates the loss of adenine from a segment of DNA, yielding a product with the mass of the full-length DNA segment minus 117.1 amu corresponding to one of the masses observed in this experiment.…”

Section: 01m Hcl Solution Samplementioning

confidence: 99%

Chemistry at the 2′ Position of Constituent Nucleotides Controls Degradation Pathways of Highly Modified Oligonucleotide Molecules

Calvitt¹,

Levin²,

Shepperd³

et al. 2010

Oligonucleotides

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A forced degradation study of a proprietary short interfering RNA (siRNA) molecule most of whose constituent nucleotides have been modified at the 2' position was conducted to assess degradation pathways and stability liabilities. The siRNA was subjected to various conditions as a solid and in solution followed by analysis with reverse-phase ultra-performance liquid chromatography-mass spectrometry. Positional isomers of degradants gave rise to multiple chromatographic peaks with identical masses. In some instances, the exact location of a modification was elucidated, but in most cases although the identity of the nucleotide affected was proposed with a high degree of confidence, its position within the oligonucleotide sequence was not determined. Reaction mechanisms were proposed for all observed major degradants based on reverse-phase ultra-performance liquid chromatography-mass spectrometry data generated in this laboratory and a search of literature sources. This work demonstrates that the chemistry at the 2' position of constituent nucleotides controls degradation pathways of highly modified siRNA molecules under various conditions and that classes of degradants can be predicted with a fair amount of confidence. A table of mass differences is presented that can be used as an aid to making partial structural assignments in oligonucleotide molecules containing similarly modified nucleotides.

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Studies on the hydrolytic stability of 2′-fluoroarabinonucleic acid (2′F-ANA)

Cited by 58 publications

References 48 publications

Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

Antisense 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (2′F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids

Chemistry at the 2′ Position of Constituent Nucleotides Controls Degradation Pathways of Highly Modified Oligonucleotide Molecules

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