Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.     

Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.     

P-chirality-dependent immune activation by phosphorothioate CpG oligodeoxynucleotides

Many of the biologic activities of phosphorothioate oligodeoxynucleotides (PS-oligos) are affected by the sense of chirality of the phosphorus atoms of the internucleotide linkages. Some of the activities are increased by the Rp stereoisomer, and others are increased by the Sp stereoisomer. In previous studies, we showed that PS-oligos containing unmethylated CpG dinucleotides in particular sequence contexts can stimulate B cells and other immune cells. These CpG PS-oligos trigger mitogenactivated protein kinase (MAPK) signaling pathways, causing the induction of B cell proliferation and cytokine and immunoglobulin secretion. We investigated whether the immune stimulation by CpG PS-oligos depends on the sense of their P-chirality. CpG PS-oligos synthesized with internucleotide phosphorothioates of Rp configuration at P-atom showed much stronger MAPK activation and induction of I kappa B degradation after 40 minutes of stimulation compared with PS-oligos synthesized with Sp linkages. In order to determine if the enhanced stimulatory effects of the Rp stereoisomer may result from differential cellular uptake, we examined the rates at which fluorescently labeled Rp or Sp CpG PS-oligos were taken up by B cells, but these were found to be identical to each other and to stereorandom PS-oligos. The stronger stimulatory effect of the R stereoisomer did not last for 48 hours, and (3)H-thymidine incorporation assays at this point showed that only the S stereoisomer was active--to approximately the same level as induced by PS-oligos with stereorandom phosphorothioate linkages. This loss of activity of the R stereoisomer most likely resulted from rapid degradation of the oligonucleotides rather than from reduced interaction with the CpG receptor because PS-oligos in which only the CpG dinucleotide was stereodefined were most stimulatory when the CpG was Rp but not when the CpG was Sp. These studies demonstrate that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs. Rp-chirality at the CpG is preferred for best stimulation at early time points, but Sp-chirality of the PS-oligo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.     

Understanding high diastereomeric discrimination in formation of oligoribonucleotide phosphorothioate linkages: the first study of pKa-dependent activation in solid-supported coupling of 2'-O-substituted ribonucleoside phosphoramidites

Activation of 2'-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.     

Understanding High Diastereomeric Discrimination in Formation of Oligoribonucleotide Phosphorothioate Linkages: The First Study of pKa-Dependent Activation in Solid-Supported Coupling of 2′-O-Substituted Ribonucleoside Phosphoramidites

Abstract

Activation of 2′-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.     

The stereochemical course of the first step of pre-mRNA splicing.

We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.     

Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscopic studies of the Rp-Rp, Sp-Sp, and Rp-Sp duplexes, [d(GGSAATTCC)]2, derived from diastereomeric O-ethyl phosphorothioates.

2D-NOE and 1H NMR chemical shift data obtained for the title oligonucleotides were compared with similar data previously reported [Broido et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified "parent" structure, [d(GGAATTCC)]2. The spectroscopically detectable structural perturbations caused by replacement of phosphate oxygen with sulfur were mostly localized within the GsA moiety, and were greater for the Rp configuration wherein sulfur is oriented into the major groove of the B-helix. UV-derived Tm measurements gave the following order of stability for the duplexes in 0.4 M NaCl: unmodified (33.9 +/- 0.1 degrees C) approximately Sp-Sp (34.1 degrees C) greater than Rp-Rp (31.7 degrees C). The title compounds were prepared by a new and convenient synthetic route which utilized HPLC to separate the diastereomeric O-ethyl phosphorothioate precursors, (Rp)- and (Sp)-d[GG(S,Et)AATTCC], for subsequent de-ethylation by ammonia in water.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.     

Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.     

Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides.

The Rp- and Sp-diastereomers of the phosphorothioate-containing oligonucleotide d[ApAp(S)ApA] have been synthesized. They and the tetramer d[ApApApA] were tested as substrates for staphylococcal nuclease, DNase II and spleen phosphodiesterase. For digestions with DNase I these oligonucleotides were converted to the 5'-phosphorylated derivates. The reactions with the nucleases were analysed by HPLC. The phosphorothioate groups of both diastereomers were resistant to the action of staphylococcal nuclease, DNase I and DNase II. While the phosphorothioate group of the Rp-diastereomer was resistant to the action of spleen phosphodiesterase, the Sp-diastereomer was hydrolysed at an estimated rate 1/100 the rate of cleavage of the unmodified tetramer. The presence of the phosphorothioate group in the center of the molecule affected the rate of hydrolysis of neighbouring phosphate groups for some enzymes. In particular, very slow release of 3'-dAMP from the Rp-diastereomer occurred on incubation with staphylococcal nuclease but the Sp-diastereomer was completely resistant. DNase II produced 3'-dAMP quite rapidly from both diastereomers of d[ApAp(S)ApA] and DNase I released 5'-dAMP from both diastereomers of d[pApAp(S)ApA] only slowly.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Photomodulation of PS-modified oligonucleotides containing azobenzene substituent at pre-selected positions in phosphate backbone

A new protocol has been developed for incorporation of a photoisomerizable azobenzene moiety into synthetic stereo-enriched [R_p] and [S_p] PS-oligonucleotides. The azobenzene pendant is attached at pre-selected positions in internucleotidic phosphorothioate oligonucleotides of both [R_p] and [S_p] diastereomers using a novel reagent, N-iodoacetyl-p-aminoazobenzene, 1. The modified oligomers are purified on HPLC, characterized by LC–MS, and examined for their thermal and photoisomerization properties. The azobenzene moiety imparts greater stability to oligomer duplexes in (E) NN configuration as compared to (Z) configuration. The placement of the azobenzene pendant close to 5′-terminus (n − 1) and 3′-terminus of the modified PS-oligos contributes maximum stability to the duplex while a gradual decline in stability occurs with azobenzene moving toward middle of the duplex. Circular Dichroism studies reveal that the chiral environment at the phosphorus center of the PS-oligos does not alter the global conformation of the DNA duplex as such, suggesting conservation of conformation of the modified DNA strands.     

Synthesis of d(GC) and d(CG) octamers containing alternating phosphorothioate linkages: effect of the phosphorothioate group on the B-Z transition

The synthesis of four oligonucleotides containing alternating phosphorothioate groups, (Rp)-and (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)- and (Sp)-d[C(p(S)GpC)p(S)G], by the phosphite approach is described. Silica gel to which 2'(3')-O-acetyluridine and 5'-succinyl groups were bound served as support for oligomer synthesis. The syntheses were carried out by dimer addition with presynthesized diastereomerically pure dinucleoside phosphorothioates as building blocks. The products were characterized by 31P NMR, nuclease P1 digestion, and oxidation to the corresponding all-phosphate-containing oligomers. The ability of each oligomer to adopt the Z conformation under high-salt conditions was screened for by circular dichroism spectroscopy. Both (Rp)-d[G(p(S)CpG)3p(S)C] and (Sp)-d[C(p(S)GpC)3p(S)G] are capable of forming Z-type structures at high NaCl concentrations. In the case of (Rp)-d[G(p(S)CpG)3p(S)C] where a phosphorothioate of the Rp configuration occurs 5' to a deoxycytidine residue, the B----Z transition is potentiated in comparison to the unmodified oligomer. (Sp)-d[G(p(S)CpG)3p(S)C] and (Rp)-d[C(p(S)GpC)3p(S)G] retain the B conformation even at high NaCl concentration.     

Hybridizing ability and nuclease resistance profile of backbone modified cationic phosphorothioate oligonucleotides

Various stereochemically pure cationic phosphorothioate oligonucleotides bearing aminoalkyl moieties were synthesized, and their duplex-forming ability against single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and triplex-forming ability against double-stranded DNA (dsDNA) were evaluated by UV melting experiments. The cationic Rp stereoisomers showed improved duplex-forming ability against ssDNA, triplex-forming ability against dsDNA and nuclease stability.     

Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.

The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined. This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases. The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease. Performing this reaction in H2 18O gave [18O]dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to [18O]dAMPS with nuclease P1. This deoxynucleoside 5'-[18O]phosphorothioate was stereospecifically converted to [18O]dATP alpha S with adenylate kinase and pyruvate kinase [Brody, R. S., & Frey, P. A. (1981) Biochemistry 20, 1245-1251]. Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms. This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus. The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state. A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate. An identical result has been previously observed with the EcoR1 endonuclease [Connolly, B. A., Eckstein, F., & Pingoud, A. (1984) J. Biol. Chem. 259, 10760-10763]. X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites. Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.     

Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes

Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of (5')diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of (5')maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of (5')maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.