Oligonucleotide Backbone Research Articles

A new approach for the synthesis of 3′-deoxy-3′- C-formyl-ribonucleosides and the synthesis of alternating methylene(methylimino) linked phosphodiester backbone oligonucleotides with 2′-OH and 2′-OMe groups

A stereoselective synthesis of 3′-deoxy-3′- C-formyl-5-methyluridine 2 is described via the dithiane 4 as the key intermediate. Compound 2 was coupled with 3 into a novel ribo-MMI dimer 1. The dimer was then incorporated into antisense oligonucleotides which were found to have high binding affinity to the target RNA.

A Short Phosphodiester Window Is Sufficient to Direct RNase H-Dependent RNA Cleavage by Antisense Peptide Nucleic Acid

The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.

Simultaneous blockage of different EGF-like growth factors results in efficient growth inhibition of human colon carcinoma xenografts.

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor alpha (TGFalpha), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFalpha, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFalpha, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFalpha, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.

Biodistribution and metabolism of a mixed backbone oligonucleotide (GEM 231) following single and multiple dose administration in mice.

Biodistribution and metabolism of a mixed backbone oligonucleotide (MBO), GEM 231, targeted to the RIalpha subunit of protein kinase A has been studied in normal and tumor xenografted mice. The study has been carried out using [35S]-labeled MBO following single and multiple administrations of doses varying from 2 to 50 mg/kg. MBO showed wide tissue distribution following intravenous and subcutaneous administration. The highest concentration of MBO was in the kidney and liver. The general disposition of MBO was followed by digitized autoradiographic pictures of tumored mice and further confirmed wide tissue disposition and also showed defined intratumor uptake of MBO. Multiple dose administration showed increased disposition in the majority of the tissues/organs, with the exception of the kidneys. Analysis of the extracted MBO by polyacrylamide gel electrophoresis (PAGE) showed the presence of primarily intact MBO along with its degraded forms. Based on our radioactivity levels, the primary route of excretion was in urine, analysis of which showed mainly degraded forms of MBO.

The impact of molecular medicine upon early cardiovascular drug development.

Heart disease remains the leading cause of death and morbidity in the western world despite significant advances in cardiovascular (CVS) therapeutics. Since the 1960s clinical pharmacologists have been very much involved in bringing forward new classes of CVS drugs. This started with the introduction of β-adrenoceptor blockers in the early 1960s and has continued with calcium antagonists, ACE inhibitors and more recently endothelin antagonists. The principles for classical drug design have been based upon interaction with: receptor sites (β-adreno-ceptor blockers), enzymes (thrombolytics and anticoagulants) and ion channels (calcium antagonists, antiarrhythmics). The advent of molecular biology has added a fourth principle for CVS drug development – that of molecular therapeutics. While classical approaches will still provide new CVS drugs (e.g. platelet IIb/IIIa receptor antagonists and directly acting thrombin inhibitors), there is now anticipation that molecular therapeutics will provide a very specific means of intervention thus potentially avoiding some of the problems associated with the non specificity of conventional approaches, e.g. side-effects associated with lack of cardioselectivity with β-adrenoceptor blockers, risk of bleeding with anticoagulants, cough with ACE inhibitors, controversy following retrospective analysis of the use of calcium antagonists. In the broader sense molecular therapeutics includes both gene therapy and oligonucleotide therapy. However the advances in genetic analysis (identification of polymorphisms associated with diseases) mean that there may be a further impact upon drug development – namely the use of known gene polymorphisms to select patient populations at risk. We will review the status of each of these and prospects for the future.

Characterization of the "helix clamp" motif of HIV-1 reverse transcriptase using MALDI-TOF MS and surface plasmon resonance.

A helix-turn-helix motif in the crystal structure of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was proposed to be a conserved nucleic acid binding domain among several nucleotide polymerizing enzymes (Hermann, T.; Meier, T.; Götte, M.; Heumann, H. Nucleic Acids Res. 1994, 22, 4625-4633). The sequence of this domain is homologous to 259KLVGKL-(X)16KLLR284 of HIV-1 RT, which acts as a "helix clamp" grasping the template-primer (T-P) complex. We characterized the helix clamp motif using MALDI-TOF MS and surface plasmon resonance (BIAcore). Our studies showed that the "helix clamp" has a nucleic acid binding function that may not be sequence specific. This evidence suggests that ionic interactions between the helix clamp and oligonucleotide backbone are not solely responsible for binding. Secondary and tertiary structures of the protein may also play a significant role in nucleic acid binding. The association and dissociation constants, ka and kd, for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 x 10(3) M(-1) s(-1) and 1.22 x 10(3) s(-1), respectively.

Real-Time Monitoring of the Hybridization Reaction: Application to the Quantification of Oligonucleotides in Biological Samples

We describe here a competitive hybridization assay using TRACE technology which can be used for real-time monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destructive, they can be sequentially repeated, thereby allowing comparison of the hybridization kinetics and binding strength of chemically modified backbone oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonucleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respective sensitivities for phosphodiester and phosphorothioate were 0.2 and 0.8 pmol/ml in plasma and 2 and 8 pmol/g in tissues, which allow to recover intact phosphorothioate sequences in some organs even after 24 h.

Vesicular stomatitis virus as model system for studies of antisense oligonucleotide translation arrest

The focus of this chapter is neither to describe the morphology and ecology of vesicular stomatitis virus (VSV) or the Rhabdoviridae nor to claim that VSV is a suitable target for antisense drug development. This chapter presents some of the characteristics of the molecular physiology of this virus and how they can be exploited to create a model system in which fundamental studies of antisense action may be studied. In vitro RNase H-mediated cleavage of an mRNA target has been demonstrated to reflect efficiency in cell culture systems in many cases. Most of the new chemistries, however, do not elicit RNase H activation. This major hurdle can be circumvented by two strategies: (1) restoring nucleolytic activity by the conjugation of suitable pendant groups or by incorporating an RNase H-competent window (mixed backbone oligonucleotide) or (2) increasing the efficiency of steric blockade. In this respect, the simple assay described in this chapter has the advantage of discriminating true translation arrest from target RNA destruction. Because natural mRNAs are used in this assay, both untranslated and coding regions can be examined. Finally, the simultaneous translation of three mRNAs provides a convenient and, important, internal control.

Oligonucleotide transport in rat and human intestine Ussing chamber models

Cellular and intestinal absorption of naked oligonucleotides (ONs) is limited and still remains a developmental challenge. A previous report in the literature suggests that ON absorption occurs via a paracellular mechanism. The aim of this study was to test this hypothesis using rat and human intestine in a Ussing chamber and in Caco-2 cells. Transport of a 35S-labelled mixed backbone ON (MBO) across human or rat intestinal tissue or across Caco-2 cells was measured after a 2-h incubation in the presence or absence of increasing MBO concentrations or with uptake inhibitors and enhancers. MBO intestinal absorption was compared with an internal standard, mannitol. 35S-MBO demonstrated very little absorption (<1%) across rat and human intestinal tissues. Transport appeared to be unsaturable up to 500 μM, and relatively insensitive to compounds that opened tight junctions or inhibited P-glycoprotein. However, preliminary studies with Caco-2 cells suggest a possible saturable mechanism at higher ON concentrations. Confocal fluorescence microscopy studies show that fluorescein isothiocyanate (FITC)-MBO was internalized into intestinal cells. Although some differences in ON transport were observed as a function of the transport model, MBO transport was mostly consistent with a transcellular, rather than a paracellular, absorption mechanism. Copyright © 1999 John Wiley & Sons, Ltd.

Oligonucleotide transport in rat and human intestine ussing chamber models.

Cellular and intestinal absorption of naked oligonucleotides (ONs) is limited and still remains a developmental challenge. A previous report in the literature suggests that ON absorption occurs via a paracellular mechanism. The aim of this study was to test this hypothesis using rat and human intestine in a Ussing chamber and in Caco-2 cells. Transport of a (35)S-labelled mixed backbone ON (MBO) across human or rat intestinal tissue or across Caco-2 cells was measured after a 2-h incubation in the presence or absence of increasing MBO concentrations or with uptake inhibitors and enhancers. MBO intestinal absorption was compared with an internal standard, mannitol. (35)S-MBO demonstrated very little absorption (<1%) across rat and human intestinal tissues. Transport appeared to be unsaturable up to 500 microM, and relatively insensitive to compounds that opened tight junctions or inhibited P-glycoprotein. However, preliminary studies with Caco-2 cells suggest a possible saturable mechanism at higher ON concentrations. Confocal fluorescence microscopy studies show that fluorescein isothiocyanate (FITC)-MBO was internalized into intestinal cells. Although some differences in ON transport were observed as a function of the transport model, MBO transport was mostly consistent with a transcellular, rather than a paracellular, absorption mechanism.

Use of the Dithiasuccinoyl (Dts) Amino Protecting Group for Solid-Phase Synthesis of Protected Peptide Nucleic Acid (PNA) Oligomers1-3

“Peptide nucleic acid” (PNA) oligomers replace the oligonucleotide backbone of DNA with an achiral and neutral poly[N-(2-aminoethyl)glycine] backbone, and the four natural nucleobases are attached through methylene carbonyl linkages to the glycine nitrogens. The present work describes the efficient conversion of Nω-Boc/side-chain Z-protected PNA monomers to the corresponding derivatives protected by the thiolyzable Nω-dithiasuccinoyl (Dts) function. After acidolytic removal of Boc, treatment with bis(ethoxythiocarbonyl) sulfide gave the Nω-ethoxythiocarbonyl (Etc) derivatives, which were silylated at the α-carboxyl and converted to the heterocycle by reaction with (chlorocarbonyl)sulfenyl chloride. Net yields of homogeneous monomers were 71−78%. Conditions in the solid-phase mode for thiolytic removal of the Dts group, and for coupling of protected monomers, have been studied extensively and optimized. A protocol featuring (i) Dts removal with dithiothreitol (DTT) (0.5 M) in acetic acid (HOAc) (0.5 M)−CH2...

Properties of mixed backbone oligonucleotides containing 3′-O-methyl ribonucleosides

Oligonucleotides containing 3′-O-methyl ribonucleosides were synthesized, and their thermal stabilities and global conformations with RNA and DNA have been studied. The duplexes displayed lower T m values as compared to the unmodified ones, and adopted A-conformations. Furthermore, they are not a substrate for RNase H, are slightly resistant to snake venom phosphodiesterase, and are not cleaved by nuclease S 1

Synthesis and enzymatic stability of oligonucleotides consisting of isonucleosides

Abstract

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.

Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells.

It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c- myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 microM but were inactive at 0.2 microM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.

Cooperative antitumor effect of mixed backbone oligonucleotides targeting protein kinase A in combination with cytotoxic drugs or biologic agents.

Cooperative antitumor effect of mixed backbone oligonucleotides targeting protein kinase A in combination with cytotoxic drugs or biologic agents.

Methods for the Characterization of Phosphatase-Stabilized 2−5A-Antisense Chimeras

We have developed chromatographic and spectrophotometric assays for determining the degree of thiolation in phosphatase-resistant 5'-monothiophosphate-capped 2-5A-antisense chimeras. Concomitantly, we have explored the reactivity of this 5'-monophosphorothioate moiety with reporter reagents such as 5-iodoacetomidofluorescein and 5,5'-dithiobis(2-nitrobenzoic acid). On the basis of these reactions, analyses for 5'-monothiophosphate-functionalized 2-5A-antisense chimeras were made possible. Kinetic experiments demonstrated that oligonucleotide backbone negative charge could retard mixed disulfide formation in the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with 5'-monothiophosphorylated 2-5A-antisense chimeras.

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A.

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Interstrand crosslinking reaction in transplatin-modified oligo-2'-O-methyl ribonucleotide-RNA hybrids.

In the context of developing an approach to irreversibly and specifically link oligonucleotides to RNA, the purpose of this work was to determine the factors interfering with the rate of the rearrangement of the transplatin 1,3-intrastrand crosslinks into interstrand crosslinks, rearrangement triggered by the formation of a double helix between platinated oligo-2'-O-methyl-ribonucleotides and their complementary strands. The rate of the rearrangement has been studied as a function of the length of the hybrids, the location of the intrastrand crosslinks, the nature of the oligonucleotide backbone, and the nature of the doublet replacing the triplet complementary to the intrastrand crosslinks. The thermal stability of the platinated hybrids has been determined in various salt conditions. The results are discussed in relation to the mechanism of the rearrangement. It is shown that the cellular proteins present weaker nonspecific interactions with single-stranded platinated oligo-2'-O-methyl-nucleotides than with the isosequential oligodeoxyribonucleotides.

Synthesis, Biophysical Properties, and Stability Studies of Mixed Backbone Oligonucleotides Containing Novel Non-Ionic Linkages

Mixed backbone oligonucleotides (MBOs) (containing ionic and non-ionic internucleotidic linkages) in which the non-ionic segments are either methylphosphotriester (PO-OMe) or primary phosphoramidate (PO-NH2) linkages have been prepared using the recently described N-pent-4-enoyl (PNT) nucleoside phosphoramidates and H-phosphonates. Biophysical properties and stability studies suggest that these MBOs are novel antisense molecules.