Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscoptc studies of theRp-Rp,Sp-Sp, andRp-Spduplexes, [d(GGsAATTCC)]2, derived from diastereomericO-ethyl phosphorothioates

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.

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“…Binding was severely reduced by the presence of the PS linkages, which goes in line with earlier reports (48,49). …”

Section: Resultssupporting

confidence: 92%

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

Moreno

Geny

Pabon

et al. 2013

Nucleic Acids Research

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“…Another mechanism to be considered is induction of interferon production such as that proposed for phosphorothioate analogs of poly-r(I-C) (21). No induction of 'yinterferon was observed in the supernatant ofthe culture with S-dC14, and 1000 units of recombinant a-or y-interferon added directly to the cultures did not inhibit the cytopathic effect in our assay system.…”

Section: Discussionmentioning

confidence: 72%

Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.

Matsukura

Shinozuka

Zon

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

482

227

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ABSTRACT- Nuclease-resistant 25 jaM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanisnm of action is likely to be complex.

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“…2 shows the 31 P NMR spectra of the purified R32S sample at 5°C (a) and 60°C (b). The 31 P signals due to normal phosphodiester bonds around 0 ppm (expansion of −6 to −1 ppm at 5°C) were well defined but the signal derived from the phosphorothioate linkage, which usually gives 31 P signals at approximately 55 ppm [44–46], was absent from the 31 P NMR spectrum taken at 5°C (a). Since the chemical analysis of the R32S sample confirmed the existence of a phosphorothioate linkage (see Section 2) and since the similarly prepared S11S substrate that contained a phosphorothioate linkage at the cleavage site showed the expected 31 P signals around 52–55 ppm (a mixture of R p and S p isomers; data not shown), we postulated that this result might have been due to broadening of the signal because of medium‐range exchanges in conformations, occurring on the NMR time scale, around the P9 phosphorothioate linkage of R32S.…”

Section: Resultsmentioning

confidence: 99%

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

Suzumura¹,

Warashina

Yoshinari

et al. 2000

FEBS Letters

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A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G 8 and A 9 , a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1 H and 31 P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the 31 P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. 31 P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S^substrate complexes generated in the absence of Mg 2+ ions were still in an inactive form and that Mg 2+ ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.z 2000 Federation of European Biochemical Societies.

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Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscoptc studies of theR_p-R_p,S_p-S_p, andR_p-S_pduplexes, [d(GG_sAATTCC)]₂, derived from diastereomericO-ethyl phosphorothioates

Cited by 85 publications

References 35 publications

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

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