Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.     

Prooligonucleotides exhibit less serum-protein binding than phosphodiester and phosphorothioate oligonucleotides

The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.     

Synthesis of chimeric oligonucleotides containing internucleosidic phosphodiester and S-pivaloylthioethyl phosphotriester residues

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.     

Enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.

Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10 <> p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpTT). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpT<>dT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpT<>dT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.     

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides.

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.     

Propynylated phosphodiester oligonucleotides inhibit ICAM-1 expression in A549 cells on electroporation

Oligodeoxynucleotides (ODN) are used largely as either primers, antisense, or triplex-forming units. Phosphodiester ODN (PO-ODN), which are very rapidly degraded by exonucleases, must be protected at their ends. Even so, their life span inside cells is quite short. Phosphorothioate ODN (PS-ODN) are less sensitive to nucleases and are extensively used as antisense. Unfortunately, unlike PO-ODN, they interact with a number of molecules, including proteins, in addition to their specific nucleic acid targets. Their affinity for their target is lower than that of PO-ODN. PS-ODN containing propyne groups on C5 of pyrimidine have been shown to have a higher affinity toward their nucleic acid target. Here, we show that propynylated PO-ODN are more stable and much more efficient than their propyne-free counterparts. They are not efficient when they are used as lipoplexes, but they act as specific antisense on electroporation.     

RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds.

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.     

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.     

Chimeric RNA with modified backbones: alternating methylene(methylimino) linked phosphodiester backbone oligonucleotides with 2'-OH and 2'-OMe groups

Synthesis of a novel ribo-MMI dimer with 2'-OH and 2'-OMe in 5'- and 3'-nucleosides, respectively is presented. The synthesis was accomplished by reductive coupling of 3'-deoxy-3'-C-formyluridine and 2'-O-methyl-5'-O-methylaminouridine via a thioacetal as the key intermediate for the top part of the dimer. Incorporation of ribo-MMI dimers into oligonucleotides increased binding affinity for target RNA.     

Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleotides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.     

The reactivity of phosphodiester bonds within linear single-stranded oligoribonucleotides is strongly dependent on the base sequence

The cleavage of short chimeric oligonucleotides containing only one reactive ribonucleoside unit, all other nucleosides being 2′-O-methylated, has been studied at pH 8.5 and 35°C. Among the 20 different sequences that did not exhibit any tendency to form a defined secondary structure, the scissile 5′-UpA-3′ and 5′-CpA-3′ phosphodiester bonds experienced >100- and up to 35-fold reactivity differences, respectively. Compared with dinucleoside monophosphates, both rate accelerations and retardations of more than one order of magnitude were observed. Even a change of a single base several nucleosides away from the scissile bond markedly affected the reaction rate. Duplex formation at the 3′- and/or 5′-side of the scissile bond was also studied and observed to be strongly rate retarding. The origin of the high sensitivity of phosphodiester bonds to the molecular environment is discussed.     

The reactivity and function of thiol groups in trout actin

1. Considerable differences were found between the rates and degrees of modification of native trout actin with iodo[2-(14)C]acetate and iodo[1-(14)C]acetamide. 2. With iodoacetate, G- and F-actin were both labelled in the N-terminal peptide only. This modification had little effect on the ability of the actin to polymerize. 3. Iodoacetamide labelled three cysteine residues in both G- and F-actin. The modified cysteine residues were identified from the position of the corresponding tryptic peptides on peptide ;maps'. 4. The modification had little effect on the ability of G-actin to polymerize, to bind ATP or to bind Ca(2+), or on the ability of F-actin to depolymerize. 5. It is concluded that the three cysteine residues present on the ;surface' of the native trout actin molecule have no direct role in the polymerization processes, the binding of ATP, or the binding of Ca(2+).