RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

RNA干涉的研究进展

生物体内导入双链RNA后会引起体内同源基因特异性的沉默,这种现象称为RNA干涉,本主要介绍RNA干涉的研究历史,作用机制和应用等方面的情况。     

Intra- and intermolecular interactions influence the reactivity of RNA oligonucleotides

The transesterification of RNA oligonucleotides was studied over a wide pH range. The rate constants obtained indicate that, under neutral conditions, oligonucleotides with an adenosine moiety as the 5'-linked nucleoside can be up to 1000-fold more reactive than the reference oligonucleotide, a 13-mer oligo-U (1). Experiments with the modified oligonucleotide with N6,N6-dimethyladenosine (9) as the 5'-linked nucleoside moiety suggest that the exocyclic amino group is involved in the reaction, influencing the reactivity of the neighboring phosphodiester bond. In addition to such intramolecular interactions, weak intermolecular interactions most probably contribute to the reactivity.     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。     

RNA干扰

RNA干扰（RNA interference,RNAi）现象是指,当与内源性mRNA编码区某段序列同源的双链RNA(dsRNA)导入细胞后,该mRNA发生特异性的降解,而导致该基因表达的沉寂。这可能反映了生物防范病毒或转座子诱导DNA突变的一种防御机制。RNA干扰已经成为一种重要的研究基因功能的有力工具,并且有希望在对疾病的防御及治疗中发挥重要的作用。     

RNA-binding proteins in RNA interference

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.     

RNA干扰抗病毒感染

RNA干扰是由双链RNA诱导的、关闭同源序列基因表达的机制。它是一种自然存在于植物、线虫、果蝇等真核细胞生物中的抵抗病毒感染方式。随着在哺乳动物细胞培养中成功地诱导RNA干扰,利用RNA干扰预防、治疗病毒感染已成为新的研究热点,并取得了有希望的成果。在未来,有望成为抗病毒感染的有效方法。     

RNA interference for antiviral therapy

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.     

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate–DNA conjugates and acridine–DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)–iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine–DNA conjugate.Electronic Supplementary Material Supplementary material is available for this article at .     

Therapeutic potential of RNA interference for neurological disorders

During the past decade, numerous molecular mediators of neurodegenerative diseases and neurological disorders have been identified and validated, yet few novel therapies have emerged and the unmet medical needs remain high. These molecular mediators belong to target classes such as ion channels, neurotransmitters and neurotransmitter receptors, cytokines, growth factors, enzymes and other proteins. In some cases, substantial pre-clinical validation exists, but the molecular target has not been readily druggable with small molecules, proteins or antibodies. RNA interference represents a therapeutic approach applicable to such non-druggable targets. Both non-viral and viral delivery strategies are being undertaken for in vivo silencing of molecular targets by RNA interference, which has resulted in robust efficacy in animal models of Alzheimer's disease, ALS, Huntington's disease, spinocerebellar ataxia, anxiety, depression, neuropathic pain, encephalitis and glioblastoma. These proof-of-concept data in animal models, together with the commencement of clinical trials using RNA interference for macular degeneration and respiratory syncytial virus infection, point to the potential of direct RNA interference for neurological disorders and neurodegenerative diseases.     

Development of strategies for conditional RNA interference

BACKGROUND: RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. METHODS: To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. RESULTS: The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. CONCLUSIONS: Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.     

Time-delayed model of RNA interference

RNA interference (RNAi) is a fundamental cellular process that inhibits gene expression through cleavage and destruction of target mRNA. It is responsible for a number of important intracellular functions, from being the first line of immune defence against pathogens to regulating development and morphogenesis. In this paper we consider a mathematical model of RNAi with particular emphasis on time delays associated with two aspects of primed amplification: binding of siRNA to aberrant RNA, and binding of siRNA to mRNA, both of which result in the expanded production of dsRNA responsible for RNA silencing. Analytical and numerical stability analyses are performed to identify regions of stability of different steady states and to determine conditions on parameters that lead to instability. Our results suggest that while the original model without time delays exhibits a bi-stability due to the presence of a hysteresis loop, under the influence of time delays, one of the two steady states with the high (default) or small (silenced) concentration of mRNA can actually lose its stability via a Hopf bifurcation. This leads to the co-existence of a stable steady state and a stable periodic orbit, which has a profound effect on the dynamics of the system.     

Vector systems of RNA interference

RNA interference is a mechanism of posttranslational (at the level of mRNA) gene silencing. Sequence-specific mRNA degradation is realized with the help of small interfering RNAs produced by processing of a precursor using Dicer, an enzyme from the RNAse III family. This mechanism is now widely used in vitro on cultures of mammalian cells in order to elucidate functions of individual genes by gene specific knockdown. Analogs of small interference RNAs are intensely expressed during embryogenesis. The mechanism of RNA interference plays an especially important role in embryogenesis of invertebrates. Identification of the functions of small noncoding RNAs is essential for understanding the genetic mechanisms underlying individual developmental stages. In order to integrate small interference RNAs in mammalian cells, various systems have been developed that allow both transient (for 48 h) and stable expression in vitro. These systems are considered in the present review.     

RNA interference in pain research

Within the course of only the last few years, RNA interference (RNAi) has been established as a standard technology for investigation of protein function and target validation. The present review summarizes recent progress made in the application of RNAi in neurosciences with special emphasis on pain research. RNAi is a straightforward method to generate loss-of-function phenotypes for any gene of interest. In mammals, silencing is induced by small interfering RNAs (siRNAs), which have been shown to surpass traditional antisense molecules. Due to its high specificity, RNAi has the potential for subtype selective silencing of even closely related genes. One of the major challenges for in vivo investigations of RNAi remains efficient delivery of siRNA molecules to the relevant tissues and cells, particularly to the central nervous system. Various examples will be given to demonstrate that intrathecal application of siRNAs is a suitable approach to analyse the function of receptors or other proteins that are hypothesized to play an important role in pain signalling. Intensive efforts are currently ongoing to solve remaining problems such as the risk of off-target effects, the stability of siRNA molecules and their efficient delivery to the CNS. RNAi has thus demonstrated that it is an extremely valuable tool for the development of new analgesic drugs.     

Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique

Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.     

RNA干扰与基因敲除

RNAi是指通过双链RNA介导特异性降解靶mRNA,导致转录后水平基因沉默的现象。其作用途径有RdRP依赖的RNAi的途径与非RdRP依赖的RNAi途径2种。利用RNAi的基因敲除技术在dsRNA序列选择、质粒或病毒为载体的dsRNA体内合成、发夹样siRNA的转录、dsRNA的导入方法等方面取得了很大进展,在研究人类或其他生物基因组中未知基因及蛋白质的功能等领域具有诱人的应用前景。     

RNA干涉与功能基因组

RNA干涉是通过双链RNA的介导特异性抑制具有相应序列的基因表达的转录后水平基因沉默机制,它为反向遗传方法研究基因功能开辟了一条新路。本综述了RNA干涉的机制以及它在功能基因组研究方面的最新进展。     

用二次电泳法研究核酸与蛋白质的相互作用

研究蛋白质与核酸的结合常遇到的问题是对蛋白质等电点及可溶度等要求较高,或难以同时处理大量标本。为克服此缺点,将待检蛋白经聚丙烯酰胺凝胶电泳后,通过洗涤去除凝胶中的ＳＤＳ,使蛋白质相对固定于凝胶中,改电泳液为ＴＡＥ或ＴＢＥ,继之用同位素标记寡核苷酸进行二次电泳,通过放射自显影直观地显现出蛋白结合核酸的结果。该法敏感,特异,对蛋白质等电点及可溶性要求低,可同时检测多个样本,值得推广使用。