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.     

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.     

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.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

A cationic derivative of amphotericin B as a novel delivery system for antisense oligonucleotides

A novel approach based on a plasma membrane permeability-disturbing agent was proposed as an antisense oligonucleotide delivery system. AMA, a derivative of the polyene antibiotic amphotericin B, formed a stable complex when mixed with phosphodiester oligodeoxynucleotides and enhanced the intracellular uptake of a 5' fluoresceinated anti-mdr1 20-mer into NIH-MDR-G185 cells. The nonlabeled phosphorothioate form of the oligodeoxynucleotide, complexed to AMA, inhibited P-glycoprotein expression with better efficiency and less nonspecific effects than when vectorized by Lipofectin. AMA may thus be a good agent for antisense strategy.     

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 .     

Inhibition of plasminogen activator inhibitor release in endothelial cell cultures by antisense oligodeoxyribonucleotides with a 5'-end lipophilic modification

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

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.     

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.     

Analysis of ribosyl-modified,mixed backbone analogs of a bcl-2/bcl-xL antisense oligonucleotide

Progress in oligonucleotide chemistry has provided second-generation antisense oligonucleotides with increased efficacy and reduced non-antisense-related toxicity. The ability of the 2'-O-(2-methoxyethylribose) (2'-MOE)-modified phosphorothioate gapmer oligonucleotide 4625, which matches the bcl-2 mRNA and has three base-mismatches to bcl-xL, to inhibit bcl-2 and bcl-xL expression and induce tumor cell apoptosis has been described. Here we investigated the consequences of adding of 2'-MOE or 2'-Me modifications to ribonucleotides at either the two ends of the sequence, or the center region together with different combinations of phosphodiester/phosphorothioate backbones on the activity of oligonucleotide 4625. The ability of the various 4625 analogs, including the parental first-generation oligonucleotide 3005, to inhibit bcl-2 and bcl-xL expression, and diminish cell growth or induce tumor cell death was assessed in SW2 lung cancer cells using real-time PCR, Western blotting and cell viability assays. Only oligonucleotide 4625 exhibited a potent bispecific antisense activity against bcl-2 and bcl-xL, which effectively reduced tumor cell viability. The other antisense oligonucleotides were either uniquely active against bcl-2 or completely inactive. Our data suggest that the 2'-MOE modification in combination with the phophorothioate gapmer chemistry is the optimal format of the 4625 sequence in terms of antisense activity and biological efficacy.     

Comparison of binding of N3'-->P5' phosphoramidate and phosphorothioate oligonucleotides to cell surface proteins of cultured cells

The binding of uniformly modified N3'-->P5' phosphoramidate and stereorandom and stereopure phosphorothioate oligonucleotides (ODN) to cell surface proteins was studied, using both a fibroblast and an epithelial cell line, to assess the effect of different analog backbone types and base composition on cell surface protein binding. Marked differences were observed, both quantitative and qualitative, in the proteins to which individual ODN bound. One phosphoramidate, antisense to the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R), bound to different proteins than did either a 6-base mismatch phosphoramidate IGF-1R sequence or a sense N-ras sequence. The latter bound poorly to the fibroblast line and predominantly to a 46 kDa protein in the epithelial line, as did many of the other ODN. This binding was not so marked as that of the isosequential end-capped phosphodiester N-ras sequence, which bound to this protein in both cell lines. Stereopure and stereorandom phosphorothioates containing a G-quartet (shown in other studies to form high-order tetrad structures), antisense to c-myc, exhibited considerable nonspecific binding to many proteins, as did the isosequential phosphoramidate. In particular, this ODN sequence gave notable binding to high molecular weight proteins. In general, binding of the c-myc ODN to proteins of 28-30, 46, 67, and 70-90 kDa was found in this study.     

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.     

bFGF反义寡核苷酸增强鼠黑色素瘤B16细胞化疗药物敏感性研究

目的:研究bFGF反义硫代寡核苷酸增强肿瘤细胞对化疗药物敏感性作用。方法:设计、合成bFGF寡核苷酸,用聚乙烯亚胺(polyemyleneimine,PEI)介导bFGF反义硫代寡核苷酸转染入黑色素瘤B16细胞,MTT法检测bFGF反义硫代寡核苷酸及其与化疗药物联合处理后的细胞增殖率;半定量RT-PCR测定bFGF反义硫代寡核苷酸转染后细胞中bFGF mRNA水平;流式细胞仪分析bFGF反义硫代寡核苷酸诱导的细胞凋亡。结果:bFGF反义硫代寡核苷酸对B16细胞增殖的抑制率为64.8%,且呈剂量依赖效应。B16细胞中bFGF mRNA被bFGF反义硫代寡核苷酸显著降低,为对照细胞的57.9%,且bFGF反义硫代寡核苷酸诱导B16细胞凋亡,凋亡率为41.8%。bFGF反义硫代寡核苷酸转染能显著增强B16细胞对阿霉素、5-氟脲嘧啶及顺铂的敏感性,非特异性硫代寡核苷酸不影响阿霉素、5-氟脲嘧啶及顺铂抑制B16细胞增殖。结论:bFGF反义硫代寡核苷酸显著增强B16细胞的化疗敏感性,表明其可协同化疗药物用于治疗肿瘤。     

Antisense Oligodeoxynucleotide Complementary to CXCR4 mRNA Block Replication of HIV-1 in COS Cells

Abstract

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatement of acquied immunodeficiency syndrome, we exmined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2μM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natual phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.     

Inhibition of human immunodeficiency virus type 1 activity in vitro by a new self-stabilized oligonucleotide with guanosine-thymidine quadruplex motifs

An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.     

Oligodeoxynucleotide analogs as informational drugs to regulate translation.

Of the chemically modified backbone analogs of oligodeoxynucleotides that have been developed for antisense applications, the phosphorothioate (PS) analog has perhaps the best properties. Nevertheless, it also has certain disadvantages, notably reduced hybridization and increased non-selective inhibition of translation, compared to the natural phosphodiester (PO) compounds. We have therefore synthesized and characterized a series of co-polymers, with the same antisense beta-globin sequence, but with different repeated sequences of PO and PS and tested them for their comparative properties. The results indicate that a PO-PS co-polymer is the best backbone modification for an antisense compound.