Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers

Kibler-Herzog, Laura; Zon, Gerald; Uznański, Bogdan; Whittier, G. L.; Wilson, W. David

doi:10.1093/nar/19.11.2979

Cited by 88 publications

(48 citation statements)

References 53 publications

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“…The 2′-position of the sugar moiety can also serve as a modification site. 2′-methoxy (2′-O-methyl) substituted oligonucleotides were found to form very stable double strands and exhibit significant nuclease resistance, which further facilitates in vivo administration [14][15][16][17][18][19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Nyakas

Stucki

Schürch

2011

J. Am. Soc. Mass Spectrom.

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Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2′-O-methyl-and methylphosphonatederivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS n ). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2′-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2′-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3′-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to w x -ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2′-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2′-O-methyl-and 2′-O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiments.

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Section: Introductionmentioning

confidence: 99%

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Nyakas

Stucki

Schürch

2011

J. Am. Soc. Mass Spectrom.

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“…First generation antisense oligonucleotides such as phosphorothioates and methylphosphonates, based on deoxyribose sugars, exist predominantly in C2′-endo conformations and prefer a B-form helix typical of DNA (7)(8)(9)(10)(11). These first generation agents generally form complexes with target RNAs that are less stable than similar complexes with unmodified DNA (7,8,(12)(13)(14).…”

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confidence: 99%

NMR Solution Structure of the N3‘ → P5‘ Phosphoramidate Duplex d(CGCGAATTCGCG)₂ by the Iterative Relaxation Matrix Approach

1998

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High-resolution 2D NMR spectra of the duplex CGCGAATTCGCG with deoxyribose sugars but with the normal phosphodiester linker replaced by an N3' --> P5' phosphoramidate (NP) group have been used to establish a solution structure for the duplex. Distance, angle, and base pair hydrogen-bonding constraints were used to refine the structure by use of the iterative relaxation matrix approach (IRMA). The phosphoramidate NH proton signal could be observed in DMSO at low temperature but not in H2O and D2O. For this reason, the structure was refined with the -NH in each of the two possible low-energy configurations. The structure with the nitrogen lone pair located between the nonbridging oxygen atoms of the 5'-phosphate group consistently had the lowest energy and RMSD values, consistent with an X-ray analysis of the same duplex [Tereshko, V., Gryaznov, S. , and Egli, M. (1998) J. Am. Chem. Soc. 120, 269-283]. In the refined structure, the sugars are in the C3'-endo conformation with the change from the normal C2'-endo conformation of deoxyribose apparently being driven by the gauche effect and the change in electronegativity from the 3'O to the 3'NH group. In agreement with preliminary studies [Ding, D., Gryaznov, S. M., Lloyd, D. H., Chandrasekaran, S., Yao, S., Ratmeyer, L., Pan, Y., and Wilson, W. D. (1996) Nucleic Acids Res. 24, 354-360], the backbone conformation in the NP duplex is very close to classical A-form values. Comparison of phosphodiester and phosphoramidate structures suggests that their backbones have global conformations that are primarily a function of the low-energy state of the sugar ring. A somewhat more complex situation arises when base pair conformation is analyzed with many of the base pairs having a conformation between those of classical A- and B-form helices. The effects of the 2' substituent are obviously important in specifying the final conformation of the stacked bases in either an A-form or B-form helix. It is clear, however, that conversion of the normal phosphodiester of DNA into a phosphoramidate linkage yields a nucleic acid that behaves much more like RNA than DNA, and it has been shown that NP sequences can bind to RNA-directed proteins [Rigl, C. T., Lloyd, D. H., Tsou, D. S., Gryaznov, S. M., and Wilson, W. D. (1997) Biochemistry 36, 650-659].

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“…Several modifications of the natural phosphodiester internucleoside bond have been introduced to improve hybridization properties of oligonucleotides as well as their cellular membrane penetration and stability and distribution inside cells (2)(3)(4). Unfortunately, the vast majority of these more hydrolytically stable analogs show reduced binding with RNA or DNA via duplex or triplex formation (5).…”

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confidence: 99%

Oligonucleotide N3'-->P5' phosphoramidates.

Gryaznov¹,

Lloyd²,

Chen³

et al. 1995

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

162

120

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Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3' -> P5' phosphoramidates containing 3'-NHP(O)(0-)0-5' internucleoside linkages and the study of Synthetic oligonucleotides have a great potential to become a new type of rationally designed therapeutic agent. These compounds could interfere with the expression of selected genes through a specific interaction with RNA or DNA regions of interest (1). Several modifications of the natural phosphodiester internucleoside bond have been introduced to improve hybridization properties of oligonucleotides as well as their cellular membrane penetration and stability and distribution inside cells (2-4). Unfortunately, the vast majority of these more hydrolytically stable analogs show reduced binding with RNA or DNA via duplex or triplex formation (5).In the present report we describe the synthesis and hybridization properties of the new analogs of nucleic acidsuniformly modified oligonucleotide N3' -* P5' phosphoramidates (Fig. 1). These compounds possess some very attractive features for therapeutic and diagnostic applications, such as a negatively charged and achiral phosphorus, good solubility in water, and phosphodiesterase digestion resistance, buttressed by a high affinity for natural RNA and DNA. MATERIALS AND METHODSOligonucleotide Synthesis. Oligodeoxyribonucleotides and oligoribonucleotides were prepared on an ABI 380B synthesizer using standard DNA or RNA assembly protocols via the phosphoramidite method. Oligonucleotide N3' -> P5' phosphoramidates were synthesized on 1 ,umol scale using an ABI 394 synthesizer and 5'-4,4'-dimethoxytrityl (DMT)-N-acyl-3'-amino-2',3'-dideoxynucleosides, as described (6). Oligonucleotides were purified by ion exchange HPLC on a Pharmacia Mono Q 10/10 column at pH 12 (10 mM NaOH) with a 1%The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. per min gradient of 1.5 M NaCl in 10 mM NaOH and a flow rate of 2 ml/min. Oligonucleotides were desalted immediately after purification on Pharmacia NAP-5 gel filtration columns and then lyophilized in vacuo.Thermal Dissociation Experiments. A Cary-Varian 1E spectrophotometer equipped with a temperature programmer and data processor from Varian was used according to the reported procedure for thermal dissociation experiments (7). Buffer concentrations and compositions are listed in the legends to Tables 1-3. Absorbance values at 260 nm were obtained at 1-min intervals with a temperature increase of 1.0°C/min. The reported tm (°C) is the temperature at the midpoint of the slope of a plot of absorbance vs. temperature.Thermodynamic Parameters Determination. Thermodynamic parameters for the dodecamers 11-13 (see T...

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Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers

Abstract: ABSTRACT

Cited by 88 publications

References 53 publications

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

NMR Solution Structure of the N3‘ → P5‘ Phosphoramidate Duplex d(CGCGAATTCGCG)₂ by the Iterative Relaxation Matrix Approach

Oligonucleotide N3'-->P5' phosphoramidates.

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Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers

Abstract: ABSTRACT

Cited by 88 publications

References 53 publications

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

NMR Solution Structure of the N3‘ → P5‘ Phosphoramidate Duplex d(CGCGAATTCGCG)2 by the Iterative Relaxation Matrix Approach

Oligonucleotide N3'-->P5' phosphoramidates.

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NMR Solution Structure of the N3‘ → P5‘ Phosphoramidate Duplex d(CGCGAATTCGCG)₂ by the Iterative Relaxation Matrix Approach