Synthesis of a N-acylsulfamide linked dinucleoside and its incorporation into an oligonucleotide.

A N-acylsulfamide linked thymidine dinucleoside was synthesized and incorporated into an oligonucleotide (ON). The interest is in a linkage analog that has a higher pKa relative to a phosphodiester and when incorporated into ONs is capable of helix formation with complementary RNA. The hybridization property of the resultant ON with RNA was shown to result in significant destabilization.

Synthesis and hybridization property of an oligonucleotide containing a 3-thioformacetal linked pentathymidylate.

A pentathymidylate fully substituted with 3'-thioformacetal intemucleotidic linkages was synthesized and subsequently incorporated into an oligonucleotide (ON) 15mer. Tm analysis was performed on the resulting ON hybridized with its complementary RNA. This duplex demonstrated slightly improved binding affinity relative to the control phosphate diester ON/RNA hybrid.

A cytosine analog that confers enhanced potency to antisense oligonucleotides.

Antisense technology is based on the ability to design potent, sequence-specific inhibitors. The G-clamp heterocycle modification, a cytosine analog that clamps on to guanine by forming an additional hydrogen bond, was rationally designed to enhance oligonucleotide/RNA hybrid affinity. A single, context-dependent substitution of a G-clamp heterocycle into a 15-mer phosphorothioate oligodeoxynucleotide (S-ON) targeting the cyclin-dependent kinase inhibitor, p27(kip1), enhanced antisense activity as compared with a previously optimized C5-propynyl-modified p27(kip1) S-ON and functionally replaced 11 C5-propynyl modifications. Dose-dependent, sequence-specific antisense inhibition was observed at nanomolar concentrations of the G-clamp S-ONs. A single nucleotide mismatch between the G-clamp S-ON and the p27(kip1) mRNA reduced the potency of the antisense ON by five-fold. A 2-base-mismatch S-ON eliminated antisense activity, confirming the sequence specificity of G-clamp-modified S-ONs. The G-clamp-substituted p27(kip1) S-ON activated RNase H-mediated cleavage and demonstrated increased in vitro binding affinity for its RNA target compared with conventional 15-mer S-ONs. Furthermore, incorporation of a single G-clamp modification into a previously optimized 20-mer phosphorothioate antisense S-ON targeting c-raf increased the potency of the S-ON 25-fold. The G-clamp heterocycle is a potent, mismatch-sensitive, automated synthesizer-compatible antisense S-ON modification that will have important applications in the elucidation of gene function, the validation of gene targets, and the development of more potent antisense-based pharmaceuticals.

Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide.

One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications have been synthesized and evaluated for enhanced cellular permeation with limited success. Phenoxazine, a tricyclic 2' deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/RNA hybrids and to enhance cellular uptake. However, the bioactivity and cellular permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimized C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide targeting SV40 large T antigen enhanced in vitro binding affinity for its RNA target and redirected RNAse H-mediated cleavage as compared with the 7-mer C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target-selective antisense activity following microinjection into cells. Incubation of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular penetration, nuclear accumulation, and subsequent antisense activity. The unique permeation properties and gene-specific antisense activity of the 7-mer phenoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-effective, self-permeable antisense therapeutics.

Synthesis and hybridization property of an oligonucleotide analog containing a 1',3'-di-O-methylene-alpha-D-fructose backbone.

Hydrogen phosphonate monomers of T (thymine) and C(m) (5-methylcytosine) bearing a 1',3'-di-O-methylene-alpha-D-fructose sugar moiety were synthesized and incorporated into an oligonucleotide. Hybridization studies by thermal denaturation experiment indicated that this oligonucleotide did not form a duplex with the complementary RNA target.

Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.

Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation both in vitro and in vivo by a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to bind to a portion of the supF mutation reporter gene. Using in vitro gel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, the in vitro binding of the c7X-TFOs was not affected, but the efficiency of in vivo triple helix formation was reduced. These results indicate the utility of the c7X substitution for in vivo gene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.

Antisense gene inhibition by C-5-substituted deoxyuridine-containing oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA and ability to inhibit gene expression is discussed. The relative binding affinity was measured by thermal denaturation (Tm) analysis, and antisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is not directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear to be major determinants of the antisense potency of the various substituted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activate cleavage of RNA by RNase H leading to potent inhibition of gene expression.

Protein engineering thrombin for optimal specificity and potency of anticoagulant activity in vivo.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

In pursuit of antisense.

The first generation of antisense oligodeoxynucleotides (ODNs) are now undergoing clinical trials, but their effects may reflect biological activities unrelated to their ability to bind RNA. Nevertheless, preclinical animal studies now suggest that phosphorothioate ODNs may be more permeable in certain animal tissues than in cell culture, raising hopes that antisense mechanisms can be exploited pharmacologically.

Nucleosides with a twist. Can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides?

The sugar moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme Northern and Southern conformations as defined in the pseudorotational cycle. In the present work, we describe how the bicyclo[3.1.0]hexane template fixes the ring pucker of 2'-deoxy-methanocarba-nucleosides 1-5 and 12 to values corresponding to either one of these two extreme conformations that are typical of nucleosides. The syntheses of the fixed Northern conformers 1-5 were performed by Mitsunobu coupling of the heterocyclic bases with the chiral carbocyclic alcohol 6 [(1R,2S,4R,5S)-1-[(benzyloxy)methyl]-2-(tert-butyloxy)-4-hydrox ybicyclo[3.1.0]hexane], while the synthesis of the Southern conformer, (S)-methanocarba-T (12), was reported earlier. Carbocyclic thymidine (carba-T, 13) was used as a reference, flexible carbocyclic nucleoside. Antiviral evaluation of these compounds revealed a very potent antiherpetic activity associated with the Northern thymidine analogue 2, which was more powerful than the reference standard acyclovir against both HSV-1 and HSV-2. (N)-Methanocarba-T (2) was further evaluated as a component of a short oligodeoxynucleotide (ODN) phosphorothioate (5'-CTTCATTTTTTCTTC-3') where all thymidines were replaced by 2. The expected thermodynamic stability resulting from the preorganization of the pseudosugar rings into a Northern conformation, typical of A-DNA, was evident by the increase in Tm of the corresponding DNA/RNA heteroduplex. However, the rigid A-tract ODN caused loss of RNase H recruitment. A detailed conformational analysis of (N)-methanocarba-T (2) and (S)-methanocarba-T (12), as representative examples of conformationally rigid pseudorotational antipodes, revealed that in addition to their different forms of ring pucker, (S)-methanocarba-T appears to be a rather stiff molecule with fewer low-energy conformational states available compared to (N)-methanocarba-T. The syn/anti-energy barrier for these nucleoside analogues is 5-6 kcal/mol higher than for common nucleosides.

Potent and selective inhibition of gene expression by an antisense heptanucleotide.

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides.

Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.

Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.

An anti-parallel triple helix motif with oligodeoxynucleotides containing 2'-deoxyguanosine and 7-deaza-2'-deoxyxanthosine.

Triple helix formation of oligodeoxynucleotides (ODNs) with a 15 base pair poly-purine DNA target in the HER2 promoter was examined by footprinting analysis. 7-deaza-2'-deoxyxanthosine (dzaX) was identified as a purine analogue of thymidine (T) which forms dzaX:A-T triplets. ODNs containing 2'-deoxyguanosine (G) and dzaX were found to form triple helices in an anti-parallel orientation, with respect to the poly-purine strand of the target DNA. In comparative studies under physiological K+ and Mg++ concentrations and at pH 7.2, the ODNs containing G and dzaX showed high affinity to the target sequence while the ODNs containing G and T were not able to bind. In the absence of added monovalent salts both ODNs showed high affinity to the target sequence. The substitution of 7-deaza-2'-deoxyguanosine for G substantially decreased the capacity of the ODNs to form triple helices under physiological conditions, indicating that dzaX may be unique in its ability to enhance triple helix formation in the anti-parallel motif.

Oligonucleotide-mediated triple helix formation using an N3-protonated deoxycytidine analog exhibiting pH-independent binding within the physiological range.

Triple helix formation with pyrimidine deoxyoligonucleotides for the sequence-specific recognition of DNA duplex targets suffers from a decrease in affinity as the pH of the medium increases to that of physiological fluids. A solution to this problem has been identified and entails the substitution of N6-methyl-8-oxo-2'-deoxyadenosine (M) for the 5-methyl-deoxycytosine base residues. The triple helix forming ability of an oligonucleotide consisting of thymidine and M residues is pH independent in the physiological range. Furthermore, M has been found to be superior to the previously used 5-methyldeoxycytidine and deoxyguanosine in conferring increased affinity for duplex DNA under physiological salt conditions.