Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides.

We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.     

Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection

A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.     

An automated algorithm for sequence confirmation of chemically modified oligonucleotides by tandem mass spectrometry

We have developed a tandem mass spectrometry (MS/MS) data analysis program for confirmation of sequence of chemically modified oligonucleotides. The method is based on the analysis of deconvoluted MS/MS data for fragment ions from three charge states and comparison of these data against a set of computer-generated masses from expected fragmentation patterns. The algorithm compares the experimental masses not only against the fragment set predicted for the expected sequence but also against a wider test set covering all next-neighbor position switches of the original sequence and all pairwise swaps of nucleosides, which in synthesis would result in molecules with masses within a preset mass tolerance. The algorithm is capable of identifying incorrect sequences that would not be distinguished by identity testing with electrospray ionization mass spectrometry. The method has been tested with permutations of the two 21-mer single strands of a chemically modified short interfering RNA containing 2′-O-methyl and phosphorothioate linkages. For both strands, challenge sequences were synthesized and tested with the premise that they were the original sequences. The algorithm correctly reported the locations of next-neighbor position switches and nucleoside swaps. The results confirm the approach as useful for MS/MS-based identity test methods for synthetic oligonucleotides.     

Stepwise synthesis of oligonucleotide–peptide conjugates containing guanidinium and lipophilic groups in their 3′-termini

Two different series of oligonucleotide–peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide–phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene.     

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of (5')diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of (5')maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of (5')maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

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.     

Synthesis and physical properties of anti-HIV antisense oligonucleotides bearing terminal lipophilic groups.

A number of phosphoramidite monomers have been prepared and used in the synthesis of antisense phosphorothioate oligonucleotides bearing 5'-polyalkyl and cholesterol moieties. Similar groups have also been attached to the 3'-end of oligonucleotides by means of functionalised CPG. Melting temperatures of duplexes formed between phosphorothioate oligonucleotides with lipophilic end-groups and complementary DNA strands were found to be identical to those formed by the equivalent unmodified phosphorothioates.     

A novel universal linker for efficient synthesis of phosphorothioate oligonucleotides

A versatile and conformationally preorganized universal linker molecule is reported here for efficient synthesis of phosphorothioate oligonucleotides. With respect to nucleoside loaded support, comparable yield and quality based on ion-pair LC-MS are obtained for both deoxy and 2'-O-methoxyethyl modified phosphorothioate oligonucleotides. No 3'-phosphate or phosphorothioate monoester or any modification of universal molecule still attached to oligonucleotide was observed. [structure: see text]     

Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro C18 column eluted with a mobile phase consisting of hexafluoro-2-propanol and triethylamine in methanol at 50 degrees C. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC-MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.     

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.     

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.     

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.     

Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity.

We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.     

A novel solid support for synthesis of oligonucleotide 3'-phosphorothioate monoesters

A new reagent immobilized on solid support allowing for solid-phase synthesis of oligonucleotides with a 3'-terminal phosphorothioate monoester is described. The support is compatible with phosphoramidite chemistry for automated oligonucleotide synthesis. Final deprotection with ammonia under standard conditions leads to oligonucleotide 3'-terminal phosphorothioate.     

Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.     

反义寡核苷酸药物癌泰得的定性分析方法研究

 癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .     

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.     

Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides

The antisense approach is conceptually simple and elegant; to design an inhibitor of a specific mRNA, one needs only to know the sequence of the targeted mRNA and an appropriately modified complementary oligonucleotide. Of the many analogs of oligodeoxynucleotides explored as antisense agents, phosphorothioate analogs have been studied the most extensively. The use of phosphorothioate oligodeoxynucleotides as antisense agents in various studies have shown promising results. However, they have also indicated that quite often, biological effects observed could be solely or partly non-specific in nature. It is becoming clear that not all phosphorothioate oligodeoxynucleotides of varying length and base composition are the same, and important consideration should be given to maintain antisense mechanisms while identifying effective antisense oligonucleotides. In this review, I have summarized the progress made in my laboratory in understanding the specificity and mechanism of actions of phosphorothioate oligonucleotides and the rationale for designing second-generation mixed-backbone oligonucleotides.