Enzymatic and hybridization properties of oligonucleotide analogs containing novel phosphoramidate internucleotide linkages

In line with the paradigm, that antisense oligonucleotides should contain minimal structural modifications, in order to minimize the risk of toxicity and antigenicity, we describe here the preparation and the properties of oligonucleotides modified to contain, in addition to phosphodiester bonds, a small number of phosphoramidate internucleotide linkages substituted with aminoethoxyethyl groups in order to convey protection against exo- and endonucleases. Prolonged stability was, in fact, found in model experiments with respective enzymes, as well as in studies done in human blood serum. Regardless of number and position of phosphoramidate linkages, the modified oligonucleotides showed only a slight decrease of Tm in hybridization studies with complementary oligonucleotides.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Oligonucleotide analogs with peptide internucleotide linkages

Oligonucleotide analogs containing one or a few glycine, L-, and D-alanine or L-and D-phenylalanine residues instead of phosphodiesterinternucleotide linkages were synthesized. The stability of the duplexes formed by modified oligonucleotides and their wildtype complements was studied. Oligonucleotides with D-alanine residues in internucleotide linkages form duplexes more stable than native ones (ΔT(m) +0.2 °C per modification), whereas other modifications destabilize the duplexes.     

Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents

Triazolyl-functionalized oligonucleotide (ON) analogs have received much attention as potential antitumor and antiviral agents. The most promising of such analogs are those exhibiting high binding affinity toward native DNA/RNA, since they may prove to be efficient antisense or siRNA agents. To date, relatively few ON analogs with triazole internucleotide linkages have been described. In this paper, we report an improved synthesis of a modified dinucleoside phosphoramidite and hybridization data of ON analogs with four-bond triazole internucleotide linkages. We believe these data are essential for comprehensive analysis of the relation between the length of triazole internucleotide linkages and duplex stability.     

Synthesis of hexanucleotide analogues containing diisopropylsilyl internucleotide linkages.

The synthesis of two silyl-linked hexanucleotide analogues is described. Hypochromicity and CD measurements indicate that the thymidine hexanucleotide analogue bears a strong resemblance to its phosphodiester-linked counterpart.     

Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages

A triester method for the synthesis of deoxynucleoside phosphorodithioate dimers is described. The phosphorodithioate linkage is introduced using a new dithiophosphorylating reagent DPSE-SP(S)Cl₂ where DPSE = 2-diphenylmethylsilylethyl. This group is removed quickly using tetra-butylammonium fluoride leading to the quantitative formation of phosphorodithioate diesters uncontaminated with the corresponding phosphorothioates. The utility of this group is demonstrated by the synthesis of a pentadecathymidylic acid, [T(PS₂)T(PO₂)]₇T, which contains alternating phosphorodithioate/phosphate diester internucleotide linkages.     

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.     

Synthesis and selective cleavage of oligodeoxyribonucleotides containing non-chiral internucleotide phosphoramidate linkages.

Oligodeoxynucleotides containing phosphoramidate internucleotide links 3'-OP(O)NH-5' have been prepared using standard solid phase phosphoramidite techniques. For the incorporation of the phosphoramidate linkages we have used monomer as well as dimer building blocks. With the monomer 3'-phosphoramidite building blocks, which are derived from 5'-amino-2',5'-dideoxynucleosides, it is possible to incorporate phosphoramidate links into specific positions within an oligodeoxynucleotide. Furthermore the synthesis of several dinucleoside phosphate derivatives which are linked by phosphoramidate bonds are described. The internucleotide phosphoramidate linkage was performed using the Staudinger reaction followed by a Michaelis-Arbuzov type transformation. After 3'-phosphitylation these dinucleosides are compatible with the current phosphoramidite methodology of oligodeoxynucleotide synthesis.     

Progress toward the synthesis of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages.

The synthesis of building blocks for the preparation of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages is described. Coupling conditions for the conversion of several of these monomers to dimers are also described.     

Immunostimulatory activity of CpG oligonucleotides containing non-ionic methylphosphonate linkages

Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG-motifs in a particular sequence context activate vertebrate immune cells. We examined the significance of negatively charged internucleoside linkages in the flanking sequences 5' and 3' to the CpG-motif on immunostimulatory activity. Cell proliferation and secretion of IL-12 and IL-6 in mouse spleen cell cultures, and spleen weights of mice increased significantly when a non-ionic linkage was placed at least four or more internucleoside linkages away from the CpG-motif in the 5'-flanking sequence. When the non-ionic linkage was placed closer than three internucleoside linkages in the 5'-flanking sequence to the CpG-motif, immunostimulatory activity was suppressed compared with that observed with the unmodified parent oligo. In general, the placement of non-ionic linkage in the 3'-flanking sequence to the CpG-motif either did not affect or slightly increased immunostimulatory activity compared with the parent oligo. These results have significance in understanding CpG oligonucleotide-receptor interactions and the development of potent immunomodulatory agents.     

Synthesis and hybridization properties of oligonucleotide analogues with novel acyclic triazole internucleotide linkages

Herein, we describe synthesis of novel acyclic dinucleotide analogues connected via triazole linkage in CuAAC reaction. Synthesis pathway starting from previously obtained building blocks containing alkyne or azide functional group is described. Further functionalization and application of dinucleotide analogues in DNA phosphoramidite solid-phase synthesis is also explained. Additionally, we have examined the influence of novel modifications on DNA duplex thermodynamic stability.     

Reactive oligonucleotide derivatives containing methylphosphonate groups. V. Increased effectiveness of complementary addressed modification of target DNA by alkylating derivatives of methylphosphonate analogs of octathymidylate containing N-(2-hydroxyethyl)phenazinium residues

Efficiency of the intracomplex alkylation of octadecadeoxyribonucleotide d(pC5A8C5) (target) by Rp- and Sp-individual diastereomers of the methylphosphonate octathymidylate 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide (-pNHCH2RCl) derivatives bearing an additional N-(2-hydroxyethyl)phenazinium residue (phn), viz. ClRCH2NHpTp.(TpTp)3TpNH(CH2)2NHPhn (I) and PhnNH(CH2)2NHpTp(TpTp)3TpNHCH2RCl (II) (p = -OP(O) (CH3)O-), has been investigated. Stabilisation of the complementary complex formed by the target oligonucleotide and methylphosphonate oligonucleotide derivatives by the Phn group considerably rose the efficiency of the intracomplex alkylation of the target as compared with alkylation by reagents without Phn. RP-isomeric derivatives of (I) and (II) proved to be the most effective alkylating reagents. Specificity of alkylation of nucleic acid target by reagents (I) and (II) is studied.     

Structure and properties of oligonucleotide analogs having phosphoramidate linkages

The oligothymidylate analogs, having several stereo regular phosphoramidate linkages, were synthesized. Melting temperatures(Tm) of complexes of the analogs and poly(dA) were measured by spectroscopic method. The abilities of the analogs to form the complexes with poly(dA) depended on their P-chirality of their modified linkages: one of the chiral isomers formed stable complexes, but another isomer formed less stable complexes.     

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.     

Cleavage properties of an oligonucleotide containing a bridged internucleotide 5'-phosphorothioate RNA linkage.

An oligonucleotide has been synthesized that contains a single bridging 5'-phosphorothioate at an RNA linkage (5'-ApCpGpGpTpCpTprCpsApCpGpApGpC-3'). This new phosphodiester linkage is found to be particularly susceptible to cleavage when compared with the corresponding oxo, deoxy and thiodeoxy derivatives. Divalent metal cations were observed to dramatically increase the cleavage rate. The products of the cleavage under a variety of conditions are a 5'-thiol-containing fragment (6mer) and a 2',3'-cyclic phosphate-containing fragment (8mer). The pseudo-first order rate constant, kobs, for cleavage at pH 7.5 (50 mM Tris-HCI) in the presence of 5 mM EDTA is 1.5 x 10(-4)/min. In the presence of 5 mM metal dichloride and 50 mM Tris-HCI, pH 7.5, the relative cleavage rate enhancements are 10, 24, 71, 98, 370 and 3400 for Mg2+, Ca2+, Mn2+, Co2+, Zn2+ and Cd2+ respectively. The rate enhancements correlate well with Pearson's HSAB principle, suggesting that cleavage is mediated in part by coordination of the metal to the 5'-mercapto leaving group. RNA linkages containing bridging 5'-phosphorothioates should prove valuable for studying the mechanistic details of a variety of RNA cleaving agents, such as ribozymes.     

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.     

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.     

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.     

Conformation and thermostability of oligonucleotide d(GGTTGGTGTGGTTGG) containing thiophosphoryl internucleotide bonds at different positions

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (TBA) is an efficient tool for the inhibition of thrombin function. We have studied conformations and thermodynamic stability of a number of modified TBA oligonucleotides containing thiophosphoryl substitution at different internucleotide sites. Using circular dichroism such modifications were found not to disrupt the antiparallel intramolecular quadruplex specific for TBA. Nevertheless, the presence of a single thiophosphoryl bond between two G-quartet planes led to a significant decrease in the quadruplex thermostability. On the contrary, modifications in each of the loop regions either stabilized an aptamer structure or did not reduce its stability. According to the thrombin time test, the aptamer with thio-modifications in both TT loops (LL11) exhibits the same antithrombin efficiency as the original TBA. This aptamer shows better stability against DNA nuclease compared to that of TBA. We conclude that such thio-modification patterns are very promising for the design of anticoagulation agents.     

Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2'-substituted 3'-deoxyribonucleosides.

In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841].