Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.     

Nucleotide positions responsible for the processivity of the reaction of exonuclease I with oligodeoxyribonucleotides.

The processive hydrolysis of single-stranded oligodeoxyribonucleotides by exonuclease I from Escherichia coli has been investigated. Oligodeoxyribonucleotides and their analogues, which contain either an abasic site or a methylphosphonate internucleotide linkage, were partially hydrolyzed by exonuclease I. The relative dissociation constant for the enzyme and each oligomeric product was calculated from the concentration of that oligomer found in solution and hence released by the enzyme before complete hydrolysis. The results have led to a characterization of the two oligodeoxyribonucleotide domains that bind to exonuclease I. The first domain, which begins at the reactive 3'-terminal phosphodiester and extends to the 7th nucleoside base, requires both phosphodiester monoanions and base residues for its interaction with the enzyme. The second domain includes phosphodiester monoanions in positions 9-13 from the 3'-terminus but does not require nucleoside bases. Methylphosphonate substitutions indicate that only two or three of these phosphodiesters, in variable positions, must remain anionic in order to obtain full enzyme binding. The residues between the two binding domains do not play a significant role in the enzyme-oligomer interaction.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Oligothymidylate analogues having stereoregular, alternating methylphosphonate/phosphodiester backbones as primers for DNA polymerase

Oligothymidylate analogues having stereoregular, alternating methylphosphonate/phosphodiester backbones, d-Tp(TpTp)4T isomers I and II and d-Tp(TpTp)3T(pT)1-5 isomers I and II, were prepared by methods analogous to the phosphotriester synthetic technique. The designations isomer I nd isomer II refer to the configuration of the methylphosphonate linkage, which is the same through each isomer. Analogues with the type I methylphosphonate configuration form very stable duplexes with poly(dA) while those with the type II configuration form either 2T:1A triplexes or 1T:1A duplexes with poly(dA) of considerably lower stabilities. The oligothymidylate analogues were tested for their ability to initiate polymerizations catalyzed by Escherichia coli DNA polymerase I or calf thymus DNA polymerase alpha on a poly(dA) template. Neither d-Tp(TpTp)4T nor d-Tp(T]Tp)3TpT served as initiators of polymerization while d-Tp(TpTp)3T(pT)2-5 showed increasing priming ability as the length of the 3'-oligothymidylate tail increased. Analogues with type I methylphosphonate configuration were more effective initiators than the type II analogues at 37 degrees C. The apparent activation energies of polymerizations initiated by d-Tp(TpTp)3T-(pT)4 and 5 isomer I were greater than those for reactions initiated by isomer II or d-(Tp)11T. The results suggest that DNA polymerase interacts with the charged phosphodiester groups of the primer molecule and may help stabilize primer/template interaction. At least two contiguous phosphodiester groups are required at the 3' end of the analogue primers in order for polymerization to occur. Interactions between the polymerase and primer also appear to occur with phosphodiester groups located at sites remote from the 3'-OH polymerization site and may be influenced by the configuration of the methylphosphonate group.     

Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage

A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 ″ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease.

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.     

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.     

Synthesis of amide-linked [(3')CH2CO-NH(5')] nucleoside analogues of small oligonucleotides

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3'-(carboxymethyl)-3'-deoxy- and 5'-amino-5'-deoxynucleoside derivatives provides amide dimers. Activated [3'-(carboxymethyl)-3'-deoxy] units with a 5'-azido-5'-deoxy function provide "masked" 5'-amino-5'-deoxy residues for chain extension, and a 5'-O-DMT-protected unit provides the 5'-terminus for attachment to a phosphodiester linkage.     

Synthesis and Properties of ApA Analogues with Shortened Phosphonate Internucleotide Linkage

A complete series of the 2 ′–5 ′ and 3 ′–5 ′ regioisomeric types of r(ApA) and 2 ′-d(ApA) analogues with the α-hydroxy-phosphonate C3 ′-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 ′-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 ′-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 ′(2 ′)-O-P-CH₂-C4 ″ linkage lacking the 5 ′-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 ′-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

Application of Oligonucleoside Methylphosphonates in the Studies on Phosphodiester Hydrolysis by Serratia Endonuclease

Abstract

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg²⁺ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.     

Effect of 2'-5' phosphodiesters on DNA transesterification by vaccinia topoisomerase

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. By introducing single 2'-5' phosphodiesters in lieu of a standard 3'-5' phosphodiester linkage, we illuminate the contributions of phosphodiester connectivity to DNA transesterification. We find that the DNA cleavage reaction was slowed by more than six orders of magnitude when a 2'-5' linkage was present at the scissile phosphodiester (CCCTT(2')p downward arrow(5')A). Thus, vaccinia topoisomerase is unable to form a DNA-(2'-phosphotyrosyl)-enzyme intermediate. We hypothesize that the altered geometry of the 2'-5' phosphodiester limits the ability of the tyrosine nucleophile to attain a requisite, presumably apical orientation with respect to the 5'-OH leaving group. A 2'-5' phosphodiester located to the 3' side of the cleavage site (CCCTTp downward arrowN(2')p(5')N) reduced the rate of transesterification by a factor of 500. In contrast, 2'-5' phosphodiesters at four other sites in the scissile strand (TpCGCCCTpT downward arrowATpTpC) and five positions in the nonscissile strand (3'-GGGpApApTpApA) had no effect on transesterification rate. The DNAs containing 2'-5' phosphodiesters were protected from digestion by exonuclease III. We found that exonuclease III was consistently arrested at positions 1 and 2 nucleotides prior to the encounter of its active site with the modified 2'-5' phosphodiester and that the 2'-5' linkage itself was poorly hydrolyzed by exonuclease III.     

Novel method of the synthesis and hybridization properties of an oligonucleotide containing non-ionic diisopropylsilyl internucleotide linkage

Efficient synthesis of a dithymidine dinucleotide analog bearing a diisopropylsilyl linkage instead of a phosphodiester linkage is described with respect to its incorporation into oligonucleotides. The diisopropylsilyl linkage was introduced into the oligonucleotide by preparation of the phosphoramidite derivative of a dithymidine dimer unit. The diisopropylsilyl-modified oligonucleotide exhibited hybridization behavior with both single strand and duplex DNA. The thermal stability of both the duplex and triplex showed a relative instability compared to the corresponding natural phosphodiester DNA, because of the steric hindrance of the isopropyl group on the silicon atom.     

Effect of phosphorothioate modifications on the ability of GTn oligodeoxynucleotides to specifically recognize single-stranded DNA-binding proteins and to affect human cancer cellular growth

We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.     

Synthesis of Amide-Linked [(3′)CH2CO-NH(5′)] Nucleoside Analogues of Small Oligonucleotides

Abstract

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3′-(carboxymethyl)-3′-deoxy- and 5′-amino-5′-deoxynucleoside derivatives provides amide dimers. Activated [3′-(carboxymethyl)-3′-deoxy] units with a 5′-azido-5′-deoxy function provide “masked” 5′-amino-5′-deoxy residues for chain extension, and a 5′-O-DMT-protected unit provides the 5′-terminus for attachment to a phosphodiester linkage.     

Synthesis of novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions and their ability to suppress human RNase L protein expression

In order to examine the effect of modifications at the 3' overhang regions of short interfering RNAs (siRNAs) on their gene-silencing activities, we designed and synthesized novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions. Suppression of human RNase L protein expression by these siRNAs was analyzed by immunoblot with RNase L-specific antibody. It was found that, at 24 h post-transfection, the modified siRNAs having the thymidine dimers with the carbamate and urea linkage suppress the protein expression 78 and 37 times more efficiently than that with the natural phosphodiester linkage, respectively. Furthermore, the siRNA containing the carbamate linkage was 37 times more resistant to nucleolytic degradation by snake venom phosphodiesterase than the siRNA consisting of the natural phosphodiester linkage. Thus, the RNA duplexes having the thymidine dimers with the carbamate or urea linkage at their 3' overhang regions will be promising candidates for novel siRNA molecules to down-regulate protein expression.     

Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.     

Oligonucleotide duplexes containing inosine, 7-deazainosine, tubercidin, nebularine and 7-deazanebularine as substrates for restriction endonucleases HindII, SalI and TaqI.

Synthetic hexadecanucleotide duplexes containing a single purine nucleotide analogue in the recognition sites of the restriction endonucleases HindII, SalI and TaqI were used to investigate the restriction site determinants required by these enzymes for sequence recognition and phosphodiester bond cleavage. The enzymes were, in general, unaffected by changes introduced into the minor groove of the helix. SalI was found to be inhibited by the major groove modifications introduced into the fourth position of its recognition sequence GTCGAC. HindII and TaqI were, by contrast, able to cleave the sites containing the analogues at this position. TaqI and, to a lesser extent, HindII could also be shown to tolerate "mismatch analogues" at this site.     

Synthesis and properties of oligonucleotide derivative with P(V) porphyrin

Oligonucleotide derivatives with a P(V)tetraphenylporphyrin at the internucleotidic phosphodiester linkage were synthesized. Their interactions with the complementary oligonucleotide and pyrene-labeled oligonucleotide were investigated. Fluorescence from the porphyrin moiety was strongly quenched by the addition of the pyrene-labeled oligonucleotide and the template oligonucleotide.     

Pyrrolidine PNA-DNA chimeric oligonucleotides with extended backbone

Cis-D-2-hydroxy-4-thymin-1-yl-pyrrolidine propionic acid unit is used to make PNA-DNA dimer block that is incorporated in DNA sequences at selected positions. Since the amide linkage is shorter than phosphodiester linkage, insertion of an extra atom in the backbone with amide linkage seems to be better accommodated for internucleotide distance-complementarity.