反义技术研究进展

反义技术利用DNA或RNA分子通过Watson Crick碱基配对原则与目的基因的mRNA互补结合 ,通过各种机制使其降解或抑制其编码蛋白的翻译 ,从而抑制目的基因的表达。与基因敲除(geneknockout)等功能缺失性研究方法相比 ,反义技术具有投入少 ,周期短 ,操作简单等优点 ,因此受到了广泛的关注。对几种常用反义技术的研究进展及存在的问题进行概述。     

反义核酸药物的研究现状

反义药物以其合理设计药物的可能性和精确的特异性广泛吸引了人们的注意,但反义药物的研究并非如人们最初预想的那样简单。本文从其特异性,稳定性,透过靶细胞的能力,作用强度,活性判定,给药途径,安全性和毒性,生产成本等诸方面对反义药物的研究现状,现存问题进行了综述。相信伴随这些问题的解决,反义药物很可能成为药典的一部分,给疾病的治疗带来益处。     

Synthesis and Duplex-Forming Property of Oligonucleotides Bearing a Novel Polyamine-Modified Intercalator at the Terminal or the Internal Position

Novel oligonucleotides bearing a polyamine-intercalator conjugate modified at the terminal or the internal position were reported. These modified oligonucleotides showed duplex-stabilization effect, and the thermodynamic analysis and the salt concentration dependency of the duplex stability revealed that the polyamine moiety also acted as the duplex stabilizer by neutralization of the phosphate negative charge.     

Inhibition of the synthesis of proteins needed for Epstein-Barr virus replication by antisense RNA against the zta gene

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication.     

β-1,3-Glucan/antisense oligonucleotide complex stabilized with phosphorothioation and its gene suppression

Most of antisense oligonucleotides (ASOs) subjected to current clinical evaluation belong to phosphorothioate (PS) analogues. Although PS has great advantage in DNase resistance, it can induce nonspecific side-effects. Thus it is important to investigate the influence of ASOs with different PS contents. In this paper, we prepared the complex consisting of schizophyllan (SPG) and ASOs attached a dA₄₀ tail with different PS contents to the 3′ end of the ODN, which is introduced to stabilize the complex with SPG. With increase of PS content in the dA₄₀, its complexation ability with SPG was improved and the complex showed high thermal stability. The thermal stability of the fully phosphorothioated ASOs was obtained by only replacing 20% of the oxygen of the phosphodiester moiety. The ability of gene suppression between PS and phosphodiester for antisense sequences was almost the same, indicating that the antisense sequences need not to be PS backbone. These data may provide new insight for the interaction between β-1,3-glucan and DNA and help to deliver therapeutic ODNs.     

Determination of the Absolute P-Configuration of a Phthalidyl-Phosphonate Thymidine-Thymidine Dimer

Abstract

Phthalidyl modified oligonucleotide thymidine-thymidine dimer building blocks were synthesized via the H-phosphonate-method. The compounds which are diastereomeric at the phosphorus atom were separated by chromatography and the absolute configuration at the phosphorus atoms was determined using ROE-experiments using the corresponding methyl-phosphonates.     

The immunostimulatory effect of IL-1β in vivo is blocked by antisense peptides complementary to the loop sequence 163-171

Antisense (AS) peptides complementary to the β-bulge surface loop VQGEESNDK (Boraschi loop) of the cytokine interleukin-1β (IL-1β) have been shown to bind IL-1β at the Boraschi loop position, and to inhibit some of the IL-1β-mediated biological effects in vitro. Here we show that primary AS peptide FVITFFSLY inhibits IL-1β-mediated immunostimulation in vivo in a dose-dependent fashion, while inactive on IL-1β-induced inflammation, an effect that takes place independently of the Boraschi loop. To the best of our knowledge, this is the first time that an AS peptide has been used successfully in vivo.     

VEGF反义寡核苷酸抑制小鼠Lewis肺癌生长的研究

目的探讨血管内皮生长因子(VEGF)反义寡核苷酸对C57BL/6小鼠Lewis肺癌肿瘤生长的抑制作用。方法制备C57BL/6小鼠皮下肺癌模型30只,随机分为3组,每组10只VEGF反义寡核苷酸(ASPODN)治疗组、VEGF正义寡核苷酸(SPODN)治疗组及对照组。接种Lewis肺癌细胞后24h内,分别皮下注射ASPODN及SPODN进行治疗,对照组只注射生理盐水,每周2次,连续4周;观察各组小鼠肿瘤的生长情况、游标卡尺测量肿瘤体积大小。所有C57BL/6小鼠于接种后第25天先用二维超声观察肿瘤的实时图象,利用脉冲多普勒获取血流频谱,获得收缩期峰值速度(PS),阻力指数(RI)。断颈处死小鼠,光镜及电镜下观察肿瘤组织形态学改变及超微结构变化,免疫组化法检测微血管密度(MVD)。结果与对照组瘤重比较(7.83±0.78)g、VEGF-ASPODN组(4.49±0.43)g能明显抑制小鼠肿瘤生长(P<0.01)、VEGF-SPODN组(7.73±0.69)g则无明显作用(P>0.05)。VEGF-ASPODN组、VEGF-SPODN组抑瘤率分别为42.7%、5.9%。组织形态学及超微结构观察,VEGF-ASPODN对肿瘤生长具有抑制作用。VEGF-ASPODN组与对照组相比较PS及RI有明显差异(P<0.01);VEGF-SPODN组与对照组相比无明显差异(P>0.05)。结论肿瘤原位注射VEGF反义寡核苷酸能抑制小鼠肺癌生长。     

mRNA靶点筛选方法研究进展

mRNA靶点筛选问题是反义核酸领域的一个难题。近年来出现了多种筛选mRNA上可接近位点以确定靶位点的方法,包括mRNA实测分析法和计算机模拟分析两大类。其中mRNA实测分析法又包括多种针对自然折叠mRNA的实验分析技术;即基因walk技术,RNaseH作图技术、寡核苷酸微阵列技术,酶作图法确定二级结构技术,核酶导向型随机RNA库位点筛选技术和随机寡核苷酸库结合逆转录位点筛选技术。这些方法在鉴定RNA可接近位点及反义核酸的设计方面均有重要作用。     

吐鲁番市郊区维吾尔族的近亲结婚率及遗传学效应

InbreedingRateandItsGeneticsEffectinUygurofTurpanAbudulaBakhy(DepartmentofBiology,XinjiangUniversity,Urumqi830046)我们分别于1993年和1994年对吐鲁番市的牙尔湖乡、葡萄沟乡和沙河子乡等郊区维吾尔族的近亲结婚情况进行了调查,并进行了遗传分析。1方法和内容调查是在当地政府和群众的帮助下,采用对各个家庭进行访问、亲眼观察并填写表格的方式进行的。调查内容包括：近亲结婚的类型,近亲结婚夫妇及非近亲夫妇结婚所生子女9岁前早亡率、白化病、全色盲、先天性聋哑等常染色体隐性遗传病以及其他疾病的发病率．2结果与讨论2…     

反义寡核苷酸抑制Fas表达用于治疗肝脏疾病

Fas(CD95)是肿瘤坏死因子受体(TNFR)家族成员。在肝脏中,Fas激活的凋亡信号可能对调节肝细胞动态平衡起作用。但在很多炎症情况下,肝脏Fas的表达水平增高。在多种临床肝病发展过程中,肝损伤与Fas表达和细胞凋亡相关。因此通过对Fas表达进行调控,从而控制肝脏中过量和异常的细胞凋亡,是一种极具潜力的保护肝脏的治疗途径。反义寡核苷酸技术已被广泛用于在许多组织中抑制特定基因的表达。用反义寡核苷酸抑制肝脏Fas表达,可以保护动物避免由细胞凋亡而造成的肝损伤以及爆发性肝炎死亡。讨论了Fas在几种肝脏疾病中的病理作用和利用反义寡核苷酸技术阻止和控制这类肝脏疾病的病变。     

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。     

大肠杆菌蛋白酶系统的研究进展

基因工程菌大肠杆菌细胞中含有多种蛋白酶,具有重要的生理功能,同时也是影响外源蛋白表达水平的重要因素,大肠杆菌蛋白酶系统的研究是工程菌改良的基础。为减少蛋白酶对外源蛋白的降解,从不同的角度做了大量工作,其中利用反义RNA技术控制蛋白酶活性是一条值得探索的思路。     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Intracerebroventricular Administration of AT1 Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II

Abstract: Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT₁) receptors in cultured cells and brain. In both liver epithelial WB and neuro-blastoma N1E-115 cells AT₁ antisense oligomers substantially decreased AT₁ receptor density, whereas angiotensin type 2 (AT₂) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT₁ antisense oligomers in rats decreased AT₁ receptor density in hypothalamic-thalamic-septal tissue, and AT₂ receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra-cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.     

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.     

Synthesis of 2′‐Substituted MMI Linked Nucleosidic Dimers: An Optimization Study in Search of High Affinity Oligonucleotides for Use in Antisense Constructs

The synthesis of a series of methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the 2′‐position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2′‐OMe MMI dimer was selected for further studies based on its synthetic accessibility and the increased thermodynamic stability conferred upon oligonucleotides incorporating this modification.