Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.     

Study of antisense oligonucleotide phosphorothioates containing segments of oligodeoxynucleotides and 2′-o- methyloligoribonucleotides

Antisense oligonucleotide phosphorothioates have been designed, which contain segments of oligodeoxynucleotide and 2′-O-methyloligoribonucleotides and studied for their biophysical and biochemical properties. Oligonucleotide phosphorothioates containing segments of 2′-O-methyloligoribonucleotides at both 3′- and 5′-ends show increased nuclease resistance, bind more strongly to complementary RNA targets, activate RNase and show increased inhibition of human immunodeficiency virus type I replication in infected cells.     

Fas基因mRNA反义靶点筛选和体外验证

应用PARASS(poly-A anchored RNA accessible sites screening) 技术筛选Fas基因mRNA 获得3个潜在反义作用靶点,靶点1、2、3分别位于Fas基因297nt-317nt、619nt-639nt和662nt-682nt。设计了对应靶点的反义寡核苷酸A1、A2、A3,和10-23型DNAzyme D1、D2和D3。将反义寡核苷酸和Fas基因RNA结合再加入RNase H进行反应,10-23型DNAzyme则直接与Fas基因RNA作用,结果表明：3个靶点的反义寡核苷酸组及DNAzyme均能降解Fas基因RNA,为有效靶点,其靶点反应优势次序为靶点3>靶点1>靶点2;而非靶点对照组和有效靶点突变了2个碱基的对照组均没有反应。靶点2和靶点3与ISIS公司经过多次实验筛选到的Fas反义作用靶点位置基本相同,表明PARASS技术的有效性和可靠性。获得的有效反义寡核苷酸和DNAzyme为后续研究打下基础。     

'Cyclicons' as hybridization-based fluorescent primer-probes: synthesis, properties and application in real-time PCR

We have studied the use of 'pseudocyclic oligonucleotides' (PCOs) (Jiang et al. Bioorg. Med. Chem. 1999, 7, 2727) as hybridization-based fluorescent probes. The resulting fluorescent tag-attached PCOs are called 'cyclicons'. Cyclicons consist of two oligonucleotides linked to each other through 3'-3' or 5'-5' ends. One of the oligos is the probe or primer-probe sequence that is complementary to a target nucleic acid (mRNA/DNA), and the other is a modifier oligo that is complementary to one of the ends of the probe oligo. A fluorescence molecule and a quencher molecule are attached at an appropriate position in the cyclicons. In the absence of the target nucleic acid, the fluorophore and the quencher are brought in close proximity to each other because of the formation of an intramolecular cyclic structure, resulting in fluorescence quenching. When the cyclicon hybridizes to the complementary target nucleic acid strand, the intramolecular cyclic structure of the cyclicon is destabilized and opened up, separating the fluorophore and quencher groups, resulting in spontaneous fluorescence emission. Fluorescent studies in the presence and absence of a target nucleic acid suggest that cyclicons exist in intramolecular cyclic structure form in the absence of the target and form the duplex with the target sequence when present. Both the cyclicons are useful for nucleic acid detection. The studies with DNA polymerase on 5'-5'-attached cyclicons suggest that the presence of quencher moiety in the probe sequence does not inhibit chain elongation by polymerase. The experiments with a 5'-5'-attached cyclicon suggest the new design serves as an efficient unimolecular primer-probe in real-time PCR experiments.     

A more efficient and specific strategy in the ablation of mRNA in Xenopus laevis using mixtures of antisense oligos.

Previously, antisense oligodeoxyribonucleotides (oligos) have been used to ablate specific mRNAs from the maternal RNA pool of Xenopus laevis oocytes. However, this strategy is limited by the dose of oligo which can be used and the fact that 100% cleavage of the target RNA is rare. Further, non-specific cleavage of other RNAs can also occur. We demonstrate that the use of several oligos against the histone H4 RNA results in a marked improvement in the efficiency of target degradation, due to synergistic action between oligos and the existence of RNA in at least two different secondary structures. We show, by using a set of overlapping oligos complementary to the entire H4 RNA, that the amount of oligo required for efficient target ablation is greatly lowered and non-specific effects are reduced.     

Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.     

A monoclonal antibody extends the half-life of an anti-HIV oligodeoxynucleotide and targets it to CD4+ cells.

An approach was sought to increase the half-life and target cell specificity of antisense oligodeoxynucleotides (oligos). A monoclonal antibody (MAb) was derived from mice immunised with an oligo complementary to a region (1-20) of the HIV genome. This MAb exerts a protective effect on the oligo from the degradation induced by plasma exonucleases in vitro and in vivo. Moreover the anti-oligo MAb dissociates from the oligo in the presence of its complementary sequence to allow hybridization of the two complementary strands. To direct the oligo to CD4+ cells the anti-oligo MAb was cross-linked to an anti-CD4 MAb. The heteroaggregate determines a 5-fold increase in the cellular membrane binding of the oligo to CD4+ lymphocytes. These findings suggest a new approach to enhancing the therapeutic action and the target specificity of antisense oligodeoxynucleotides useful for the selective inhibition of HIV replication in vivo.     

Molecular Recognition Theory of the complementary (antisense) peptide interactions

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203–207; Nature Med. 1 (1995) 876–878; Biochem. J. 234 (1986) 679–683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5′→3′ direction) and antisense peptides coded in 3′→5′ direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide–receptor systems with verified ligand–receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an “in vivo” experiment. It was shown that 3′→5′ peptide antisense of -MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.     

Accessible 5'-end of CpG-containing phosphorothioate oligodeoxynucleotides is essential for immunostimulatory activity

In our ongoing efforts to decipher the sequence and structural requirements in the flanking region of the CpG motif in phosphorothioate oligodeoxynucleotides (PS-oligos), we have examined the requirement of free 5'- and 3'-ends of PS-oligos on immune stimulation. Our model studies using 3'-3'-linked (containing two free 5'-ends) and 5'-5'-linked (containing two free 3'-ends) CpG-containing PS-oligos demonstrate that immunostimulatory activity is significantly reduced when the 5'-end of the PS-oligo is not accessible, rather than the 3'-end, suggesting that the 5'-end plays a critical role in immunostimulatory activity.     

RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity.

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.     

Comparison of anti-RNA properties of normal and ellipticine functionalized alpha and beta-oligonucleotides.

RNA is not cleaved as a consequence of the binding of RNase H to the duplex between RNA and a complementary alpha-oligodeoxyribonucleotide (oligo). In consequence targets have been selected which do not a priori require the action of RNase H to inhibit genetic expression. Two models have been used: The Friend Murine Leukemia Virus (F-MuLV) and the synthesis of rabbit beta globin.alpha-oligos trigger specific inhibitions in both systems. The functionalisation in 5' with the intercalating agent 9-NH2-ellipticine renders the oligos resistant to degradation and allows a direct action on cells.     

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.     

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.     

Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments.

We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2'-5'linkages at each end) with the complementary 3'-5'-DNA and -RNA target strands suggest that 2'-5'-ribonucleoside incorporation into 3'-5'-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3'-5'-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2'-5'linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2'-5'modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3'-5'-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3'-5'linkages. The current results suggest that a limited number of 2'-5'linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents.     

A scientific journey through the 2-5A/RNase L system

The antiviral and antitumor actions of interferons are caused, in part, by a remarkable regulated RNA cleavage pathway known as the 2-5A/RNase L system. 2′-5′ linked oligoadenylates (2-5A) are produced from ATP by interferon-inducible synthetases. 2-5A activates pre-existing RNase L, resulting in the cleavage of RNAs within single-stranded regions. Activation of RNase L by 2-5A leads to an antiviral response, although precisely how this happens is a subject of ongoing investigations. Recently, RNase L was identified as the hereditary prostate cancer 1 gene. That finding has led to the discovery of a novel human retrovirus, XMRV. My scientific journey through the 2-5A system recounts some of the highlights of these efforts. Knowledge gained from studies on the 2-5A system could have an impact on development of therapies for important viral pathogens and cancer.     

细菌中RNase HI介导RNA降解的研究进展

在细菌细胞中,为了维持基因组稳定和正常的生命活动,RNase HI通常以降解RNA/DNA杂合链中RNA的方式来防止复制中引物的积累以及转录中R环的形成。RNase HI对底物的识别主要依赖于DNA与RNA结合槽,对底物的催化主要依赖于DEDD基序和位于活性位点附近柔性环中的一个组氨酸。以Mg²⁺为代表的金属离子在催化过程中发挥了至关重要的作用。杂交双链中ssDNA突出部分的类型决定了RNase HI的作用模式：在没有突出或在ssDNA的5′端存在突出部分的情况下,RNase HI作为一种非序列特异性核酸内切酶随机地降解RNA;当ssDNA的3′端存在突出部分时,RNase HI依靠5′核酸外切酶活性对RNA进行连续切割。RNase HI、Rep、DinG和UvrD通过与单链DNA结合蛋白(single-stranded DNA-binding protein, SSB)的C端尾部的6个残基相互作用被招募到复制叉附近,并可能以协作的方式解决复制-转录冲突。RNaseHI的缺失或活性降低将引起DNA结构不稳定、基因突变、转录装置回溯和复制不协调等一系列有害后果。RN...     

Nucleotide pyrophosphatase/phosphodiesterase 1 is responsible for degradation of antisense phosphorothioate oligonucleotides

The rapid degradation of unmodified phosphodiester oligodeoxynucleotides (PO-oligos) by exo -and endonucleases limits their application as antisense constructs and requires the synthesis and use of modified oligonucleotides. Phosphorothioate analogs of oligonucleotides (PS-oligos) are much more stable against nucleolytic degradation than their unmodified counterparts, and this is one of the reasons for which they are a promising class of antisense oligonucleotides. However, PS-oligos also undergo slow hydrolysis by enzymes present in plasma. The oligonucleotide degradation proceeds mainly from the 3' -end, resulting in the formation of a typical ladder of shorter products and the release of the mononucleoside 5' -phosphorothioates. So far, little has been known concerning the molecular identity of the enzymes involved in the degradation of PS-oligos. We now identify the human plasma 3' -exonuclease responsible for their degradation as a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9), also known as the plasma cell differentiation antigen PC-1. We also show that adenosine or deoxyadenosine (alpha-thio)triphosphates can act as potent inhibitors of NPPs.     

Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation

Antisense oligodeoxynucleotides (oligos) are widely used for functional studies of both prokaryotic and eukaryotic genes. However, the identification of effective target sites is a major issue in antisense applications. Here, we study a number of thermodynamic and structural parameters that may affect the potency of antisense inhibition. We develop a cell-free assay for rapid oligo screening. This assay is used for measuring the expression of Escherichia coli lacZ, the antisense target for experimental testing and validation. Based on a training set of 18 oligos, we found that structural accessibility predicted by local folding of the target mRNA is the most important predictor for antisense activity. This finding was further confirmed by a direct validation study. In this study, a set of 10 oligos was designed to target accessible sites, and another set of 10 oligos was selected to target inaccessible sites. Seven of the 10 oligos for accessible sites were found to be effective (>50% inhibition), but none of the oligos for inaccessible sites was effective. The difference in the antisense activity between the two sets of oligos was statistically significant. We also found that the predictability of antisense activity by target accessibility was greatly improved for oligos targeted to the regions upstream of the end of the active domain for beta-galactosidase, the protein encoded by lacZ. The combination of the structure-based antisense design and extension of the lacZ assay to include gene fusions will be applicable to high-throughput gene functional screening, and to the identification of new drug targets in pathogenic microbes. Design tools are available through the Sfold Web server at http://sfold.wadsworth.org.