Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site

Connolly, Bernard A.; Potter, Barry V. L.; Eckstein, Fritz; Pingoud, Alfred; Grotjahn, Lutz

doi:10.1021/bi00310a010

Cited by 170 publications

(108 citation statements)

References 42 publications

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“…Many examples (17,19,30,61) have shown that R p -oligonucleotide phosphorothioates elute before the S p on reverse phase columns using triethylammonium acetate buffers. This was found here in every case, and the faster elution of R p -isomers was also maintained when the buffer was altered to morpholine acetate.…”

Section: Synthesis Of Phosphorothioate-containing Oligonucleotides-mentioning

confidence: 99%

“…These stereospecificities are reversed with nuclease P1 (63). Therefore the phosphorothioates were separately digested with nuclease P1 and snake venom phosphodiesterase and the resulting deoxynucleoside products were analyzed by reverse phase HPLC (17,30). When "early" isomers were treated with the snake venom enzyme, a peak corresponding to dNMPs was observed by HPLC, whereas reaction with P1 gave a NsN unhydrolyzed dinucleotide peak (not shown).…”

Section: Synthesis Of Phosphorothioate-containing Oligonucleotides-mentioning

confidence: 99%

“…This has been widely carried out for protein amino acids using site-directed mutagenesis and for DNA with modified bases (11)(12)(13)(14)(15)(16). For DNA phosphates the most useful analogues are the phosphorothioates (17)(18)(19)(20)(21), illustrated in Fig. 1, which exist as a pair of diastereomers.…”

mentioning

confidence: 99%

“…Despite these differences, the single known structure of a phosphorothioate-containing oligonucleotide shows minimal structural perturbations as compared to the all-phosphate parent (28). Oligonucleotide phosphorothioates have been used to determine the stereochemical course of the reactions catalyzed by EcoRI (29) and EcoRV (30) and to probe details of protein-DNA interactions (31)(32)(33)(34).…”

mentioning

confidence: 99%

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Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Thorogood

Grasby

Connolly

1996

Journal of Biological Chemistry

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Section: Synthesis Of Phosphorothioate-containing Oligonucleotides-mentioning

confidence: 99%

Section: Synthesis Of Phosphorothioate-containing Oligonucleotides-mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Thorogood

Grasby

Connolly

1996

Journal of Biological Chemistry

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“…These studies have been extended to investigate reaction mechanisms involving restriction enzymes such as EcoRI (52) and EcoRV (53), DNA integration (54), and site-specific DNA recombination (55). All of these reactions proceed with an inversion of configuration as a result of a single nucleophilic substitution.…”

Section: Phosphorothioatesmentioning

confidence: 99%

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

Verma

Eckstein²

1998

Annu. Rev. Biochem.

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303

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Synthetic oligonucleotide analogs have greatly aided our understanding of several biochemical processes. Efficient solid-phase and enzyme-assisted synthetic methods and the availability of modified base analogs have added to the utility of such oligonucleotides. In this review, we discuss the applications of synthetic oligonucleotides that contain backbone, base, and sugar modifications to investigate the mechanism and stereochemical aspects of biochemical reactions. We also discuss interference mapping of nucleic acid-protein interactions; spectroscopic analysis of biochemical reactions and nucleic acid structures; and nucleic acid cross-linking studies.The automation of oligonucleotide synthesis, the development of versatile phosphoramidite reagents, and efficient scale-up have expanded the application of modified oligonucleotides to diverse areas of fundamental and applied biological research. Numerous reports have covered oligonucleotides for which modifications have been made of the phosphodiester backbone, of the purine and pyrimidine heterocyclic bases, and of the sugar moiety; these modifications serve as structural and mechanistic probes. In this chapter, we review the range, scope, and practical utility of such chemically modified oligonucleotides. Because of space limitations, we discuss only those oligonucleotides that contain phosphate and phosphate analogs as internucleotidic linkages. CONTENTS

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Oxathiaphospholane Approach to the Synthesis of P-Chiral, Isotopomeric Deoxy(ribonucleoside phosphorothioate)s and Phosphates Labeled with an Oxygen Isotope

2001

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Introduced by Eckstein, phosphorothioate analogues of nucleotides have become an indispensable tool for studying the metabolism of nucleic acids. [1] Standard chemical methods for the synthesis of oligo(deoxyribonucleoside phosphorothioate)s (PS-Oligos) provide a mixture of 2 n diastereoisomers, where n is the number of phosphorothioate linkages. [2] The enzymatic synthesis of stereodefined PS-Oligos is limited to the preparation of (all-R P )-oligomers because of the stereoselectivity of available DNA and RNA polymerases. The first method for stereocontrolled chemical synthesis of PS-Oligos was elaborated in our group, [3] and several alternative methods were recently reported. [4,5] Stereodefined PS-Oligos were used for studying the mode of action of several bacterial and human enzymes [6±8] and the stereodependent avidity of PS-Oligos toward complementary DNA or RNA. [9] However, the presence of a sulfur atom affects the properties of internucleotide bonds, mostly due to the different steric requirements of sulfur atoms (PÀS vs PÀO bond length), different affinity towards metal ions, and changes in the distribution of the negative charge in the phosphorothioate anion.[10] Therefore, the hydration pattern of PS-Oligos is different from that of natural oligonucleotides, [11] and this obstructs the evaluation of kinetic data of ªrescue effectsº of thiophilic metal ions, and makes analysis of direct or watermediated contacts between metal ions and phosphate groups much more difficult. These inconveniences could be avoided by using P-chiral isotopomeric phosphates. [12] Here we describe the synthesis of stereodefined oligo(deoxyribonucleoside [ 18 O]phosphorothioate)s (PS 18 O-Oligos) and oligo-(deoxyribonucleoside [ 18 O]phosphate)s (P 18 O-Oligos), in which both of the nonbridging oxygen atoms of the internucleotide bond were replaced by S and 18 O, or one of them was replaced by 18 O, respectively. Oligonucleotides containing a single P-chiral [ 16 O, 18 O] internucleotide bond were first used by Eckstein [13] in studies on Eco RI endonuclease. Stereodefined P 18 O-Oligos can be used to investigate the interaction of particular oxygen atoms with other molecules or metal ions, given analytical methods that allow the isotopic effect to be measured with satisfactory accuracy. [14]

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Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site

Cited by 170 publications

References 42 publications

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

Oxathiaphospholane Approach to the Synthesis of P-Chiral, Isotopomeric Deoxy(ribonucleoside phosphorothioate)s and Phosphates Labeled with an Oxygen Isotope

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