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.     

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.     

Oligothymidylates covalently linked to an acridine derivative and with modified phosphodiester backbone: circular dichroism studies of their interactions with complementary sequences.

Oligothymidylates involving alternating alkyl phosphotriester-phosphodiester or methylphosphonate-phosphodiester backbones and covalently linked to an acridine derivative have been studied using circular dichroism. Two isomers with the same diastereoisomeric configuration for all the phosphotriesters (ethyl triester and neopentyl triester) or the methylphosphonate linkages were studied. These six compounds were compared to the parent oligonucleotide with unmodified phosphodiester bonds. Intramolecular interactions between the acridine and the bases of the oligonucleotides were revealed by the induced circular dichroism of the acridine dye. Binding to poly(rA) and poly(dA) induced large changes in the circular dichroism signal. All oligothymidylates formed double-stranded complexes with poly(rA). Substitution of phosphotriesters and methylphosphonates to phosphates allowed both double- and triple-stranded structures to be formed with with poly(dA). The double-stranded structures formed with poly(rA) and poly(dA) were characterized by different environments of the acridine dye. The circular dichroism spectra of the complexes with poly(dA) and the thermal stabilities of the complexes formed with both poly(rA) and poly(dA) were drastically dependent of the diastereoisomeric configuration of the phosphate modification. For the complexes formed with the pseudoequatorial stereoisomer the modification of the phosphate groups increased the stability of the complexes as compared with the oligothymidylate containing only phosphodiester linkages whereas it decreased it for pseudoaxial modifications.     

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.     

Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.     

Reactivity of oligonucleotide derivatives, containing methylphosphonate groups. VI. Increase in the effectiveness of directed action of alkylating derivatives of oligonucleotide methylphosphonate analogs on nucleic acids in the presence of effectors--3',5'-bis-N-(2-hydroxyethyl)-phenazine derivatives of oligonucleotides

Effectors for increasing the efficiency of DNA modification with the alkylating methylphosphonate analogues of oligodeoxyribonucleotides (MFAO) were suggested. Oligodeoxyribonucleotide d(pC5A8ACAATG) used as a target DNA treated with alkylating derivatives of octathymidylate having alternating methylphosphonate and phosphodiester internucleotide bonds (both Rp- and Sp-individual diastereoisomers of MFAO were used) and bearing alkylating 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide residue at the 3'-end. The reactions were carried out in the presence of an effector, hexadeoxyribonucleotide derivative PhnNH(CH2)2NHpCATTGTpNH(CH2)2NHPhn bearing two N-(2-hydroxyethyl)phenazinium (Phn) residues at the 3'- and 5'-ends and being complementary to the part of the target DNA neighbouring with octaadenylate. It was shown that Tm of the duplex formed by the target DNA, octathymidylate and effector is by 7-13 degrees C higher than in the absence of the effector, thus considerably increasing the efficiency of the intracomplex alkylation of the target (e.g., at 40 degrees C, the increase for the reagent based on the Rp-isomer is sixfold). Specificity of the target DNA modification by the MFAO alkylating derivatives in the presence of effector is same as with reagents based on oligodeoxyribonucleotides with natural internucleotide bonds.     

Preparation and properties of a new type of acyclic, achiral nucleoside analogue

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by Tm measurements showing a deltaTm of -2 to -6.5 degrees C per modification. An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage by a 3'-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues.

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.     

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.     

Reactivity of oligonucleotide derivatives, containing a methylphosphate group. Affinity modification of target DNA by 4-N-(methyl-N-2-chlorethylamino)benzyl 3'- and 5'-phosphamide derivatives of octathymidylate containing methylphosphonate residues

Modification of 5'-32P-labelled octadecadeoxyribonucleotide d(pC5A8C5) (III) with octathymidylate methylphosphonate derivatives bearing both 3'- and 5'-terminal alkylating 4-(N-2-chloroethyl-N-methylamino)benzylphosphoamide residue has been investigated. Yield in the modification depends on configuration of methylphosphonate fragment, in case of Rp-isomer it may amount to 90%. Specificity of alkylation of nucleic acide target (III) by reagents based on the oligonucleotide methylphosphonates is almost the same as by reagents based on the oligonucleotides having phosphodiester internucleotide bonds.     

Interactions between single-stranded DNA binding protein and oligonucleotide analogs with different backbone chemistries

Chemical modification of backbone structures has been an important strategy in designing oligonucleotides capable of improved antisense effects. However, altered backbone chemistry may also affect the binding of oligonucleotides to key cellular proteins, and thus may impact on the overall biological action of antisense agents. In this study we have examined the binding of oligonucleotides having four different backbone chemistries to single-strand binding protein (SSB), a protein having a key role in DNA repair and replication. The oligomers tested had the same sequence, while the internucleoside linkages were phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), or methylphosphonate (MP). We found that both PS and PS2 oligomers bound to SSB with higher affinity than PO oligonucleotides, while MP oligonucleotides did not bind appreciably at the concentrations tested. Oligonucleotide length was also an important factor in binding to SSB, but sequence was less critical. These observations indicate that backbone chemistry is an important factor in interactions between oligonucleotides and critical cellular proteins, and thus may be a key determinant of the biological effects of antisense oligonucleotides. © 1997 John Wiley & Sons, Ltd.     

Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.

A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.     

Modified oligonucleotides in rabbit reticulocytes: uptake, stability and antisense properties.

We have investigated the behaviour of antisense oligonucleotides in rabbit reticulocytes. Both backbone-modified oligomers (methyl-phosphonate and phosphorothioate analogues), anomers of nucleotide units (alpha) and oligonucleotides linked to various ligands (intercalator, polylysine, dodecanol) were tested with respect to cellular uptake and inhibition of protein synthesis. Oligonucleotides added at an external concentration of 10 microM slowly entered the cell up to a concentration of a few hundred nanomolars. A large fraction of phosphorothioate analogues was found to be associated with the membrane. alpha-, methylphosphonate and phosphorothioate analogues remained intact during the incubation with reticulocytes although the latter were partly dephosphorylated. Antisense oligonucleotides were targeted against three different sites of the rabbit beta-globin mRNA: the 5' end of the message, the initiator AUG or the coding sequence. No specific effect on beta-globin synthesis was observed with any of the investigated compounds.     

Oligonucleotide N3'-->P5' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3'-->P5' phosphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with complementary native phosphodiester DNA and exceptionally stable duplexes with RNA strands. Increases in duplex melting temperature, deltaTm, relatively to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modified nucleoside. Moreover, the phosphoramidate compounds form extremely stable triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of these triplexes usually exceed that of the isosequential phosphodiester counterparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nucleases both in vitro and in vivo. Oligonucleotide phosphoramidates apparently are cell permeable, and they have a good bioavailability and biodistribution, while being non-toxic in mice at therapeutically relevant doses. Duplexes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these compounds exerted high sequence-specific antisense activity in various cell lines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biological activity of these compounds relative to RNase H inducing oligonucleotides. Experimental results also indicate that oligonucleotide phosphoramidates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidate double stranded complexes can structurally mimic native RNA complexes, which could be efficiently and specifically recognized by the RNA binding proteins, such as HIV-1 Rev and Tat.     

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.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.