Synthesis of Spirannic Nucleosides and Their Incorporation into Oligonucleotides

Abstract

The spirannic nucleosides dH _α, dH _β, sB and cB were synthesized and incorporated into oligonucleotides using the cpg-oxalyl solid support strategy.     

Disaccharide Nucleosides And Their Enzymatic And Chemical Incorporation Into Oligonucleotides

Abstract

A high yield synthesis of different O-ribofuranosylnucleosides has been achieved. Kinetics of the acid-catalysed hydrolysis of disaccharide nucleosides has been studied. Chemical and enzymatic incorporation of 2′-O-ribofuranosyl-nucleoside residue into oligonucleotides was investigated.     

Synthesis of Fully Protected Nucleoside- Folic Acid Conjugated Phosphoramidites and Their Incorporation into Antisense Oligonuleotides

Abstract

A novel synthesis of the nucleoside-folic acid conjugates has been accomplished. This approach allowed us to synthesize several analogs, which were converted to phosphoramidites and successfully incorporated into therapeutically active antisense oligonucleotides.     

反义寡核苷酸的化学修饰

反义寡核苷酸的功能在很大程度上取决于其稳定性、生物利用度及与靶基因结合或反应的特性．通过特定的化学修饰可以改变这些物理、化学特性,从而增加其抗病毒、抗肿瘤及对其他特定基因的表达抑制活性．     

Synthesis of Novel Pyrene-Bearing C-Nucleoside and Its Incorporation into Oligonucleotides

Abstract

Phosphoramidite of (1,2,4-triazol-1-yl)-4-[(4-pyren-1-ylbutyl)amino]-5-(2-O-methyl-5-dimethoxytrityl-β-D-ribofuranosyl)pyrimidine has been synthesized, incorporated into polypyrimidine oligoaucleotides, and studied for thier triplex forming capacity.     

Incorporation of Glucoamylase into Some Polyelectrolyte Complexes

The major wax component secreted by Anomoneura mori Schwarz nymph was identified as lacceryl laccerate, n-dotriacontyl n-dotriacontanoate. Its content in the wax was 93.8 %.     

Equal Incorporation of Both Parental Bacteriophage T7 Deoxyribonucleic Acid Strands into Intracellular Concatemeric Deoxyribonucleic Acid

After infection of Escherichia coli B with radiolabeled T7 bacteriophage, the parental deoxyribonucleic acid label was found in both polynucleotide chains of the intracellular T7 concatemer.     

反义寡核苷酸的非反义作用

反义核酸作为一种调控特定基因表达的手段,是通过与特异ｍＲＮＡ分子上５′翻译区、ｍＲＮＡ启动－编码重叠区、核内ｈｎＲＮＡ剪接供点结合抑制ｍＲＮＡ的翻译、剪接、转运或通过形成ＲＮＡ－ＤＮＡ杂合双链,激活ＲＮａｓｅＨ,降解ｍＲＮＡ来达到抑制靶基因表达的目的...     

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。     

Chemical Incorporation of 1-Methyladenosine,Minor tRNA Component,into Oligonucleotides

Abstract

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.     

Conversion of Unprotected Amino-Link Oligonucleotides into Their Tetramethylguanidinium Derivatives

Abstract

The preparation of tetramethylguanidinium oligodeoxynucleotide (ODN) derivatives by reaction of the corresponding aminoalkyl-ODN with the uronium salts HBTU, TBTU or HATU, respectively, is described. The binding affinity of the new tetramethylguanidinium ODN derivatives was determined.     

反义寡核苷酸递送方法研究进展

如何将反义寡核苷酸 (AS ODNs)有效递送进入细胞是反义核酸领域面临的一大难题。近年来 ,出现了多种寡核苷酸 (ODNs)的递送方法。在培养细胞中 ,使用的递送方法包括阳离子载体包裹、特异受体的配体导向、ODNs偶联修饰、细胞膜辅助穿透以及利用逆转录病毒载体转染等 ,其应用有效增强了AS ODNs的作用效果 ,大幅度降低了AS ODNs的使用浓度 ;在体内 ,由于临床使用裸露AS ODNs连续给药能达到一定的反义效果 ,而使递送方法的研究和应用尚处于初步尝试和探索之中 ,迄今报道的递送方法有脂类和非脂类两类。     

反义寡核苷酸prop5在A549细胞内具有抗流感病毒活性

目的：研究硫代反义寡核苷酸prop5在细胞水平的抗流感活性及其作用机制。方法：cy3标记prop5用于考查人肺腺癌细胞A549对硫代反义寡核酸的摄取;利用实时荧光定量PCR检测流感病毒RNA拷贝数,Western印迹检测prop5对PDCD5蛋白表达和caspase-3蛋白剪切的抑制;利用间接免疫荧光和Western印迹检测prop5对病毒核糖核蛋白复合体（RNP）出核的影响;利用TUNEL检测prop5对流感病毒引起细胞凋亡的抑制作用。结果：流感病毒感染促进A549细胞摄取prop5;prop5下调感染病毒的A549细胞中PDCD5蛋白的表达,并能抑制流感病毒的复制;prop5抑制流感病毒引起的A549细胞的凋亡;prop5抑制病毒RNP出核。结论：prop5在细胞水平具有抗流感病毒活性,其作用机制可能同抑制RNP出核有关;本研究为进一步探讨宿主-病毒相互作用和抗流感药物开发奠定了基础。     

Penetration of Bdellovibrio bacteriovorus into Host Cells

Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.     

Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.     

Tubulin Dimers Oligomerize before Their Incorporation into Microtubules

In the presence of GTP, purified dimers of α- and β-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.     

Effects of Calmodulin Antisense Oligonucleotides on Chemoresponse in Paramecium

The calcium/calmodulin-regulated Ca-ATPase of the plasma membraneis implicated in Paramecium chemosensory transduction. Calmodulinantisense oligonucleotides electroporated into Paramecium disruptchemosensory responses to sodium acetate but not to ammoniumchloride. Chem. Senses 21: 55–58, 1996.     

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.     

In Vivo Delivery of Antisense Oligodeoxynucleotides into Rat Kupffer Cells

Abstract

Kupffer cells play a key role in the pathogenesis of liver diseases. Liver injury is believed to result from an excessive release of cytokines and prostanoids from these cells. A targeted delivery of antisense oligonucleotides into Kupffer cells might reduce or prevent liver injury. In this report, we describe a method in which anionic liposome-encapsulated antisense phosphorothioate oligodeoxynucleotides (S-Oligos) are delivered to Kupffer cells in vivo. Delivery was assessed using an antisense S-Oligo (TJU-2749) targeted against the 3’ untranslated region of rat tumor necrosis factor-α mRNA. At 90 min post-intravenous injection, 90% of the S-Oligo was absorbed from circulation. Of this, 40% was found in the liver and 10% in spleen. Other organs, including lungs, kidneys, muscle, stomach, brain, testes and small intestine, showed only minor incorporation (<5%). Greater than 65% of the liver-associated S-Oligo was found in Kupffer cells. Relative accumulation of S-Oligo in Kupffer cells was 200-fold that of the combined body tissues. For an average injected dose of 1.2 mg antisense/Kg body weight, the intracellular concentration of the S-Oligo attained in Kupffer cells was 65 μM. These studies suggest that liposome-encapsulated delivery provides an efficient means of targeting antisense molecules to Kupffer cells in vivo.