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维基百科，自由的百科全书
慢病毒会设计shRNA并注入到哺乳动物细胞中，进行RNA干扰。
RNAi示意图

RNA干扰（RNA interference，缩写为RNAi）是指一种分子生物学上由双链RNA诱发的基因沉默现象，其机制是通过阻碍特定基因的转译或转录来抑制基因表达。当细胞中导入与内源性mRNA编码区同源的双链RNA时，该mRNA发生降解而导致基因表达沉默[1]。与其它基因沉默现象不同的是，在植物和线虫中，RNAi具有传递性，可在细胞之间传播，此现象被称作系统性RNA干扰（systemic RNAi）[2][3]。在秀丽隐杆线虫上实验时还可使子一代产生基因突变，甚至于可用喂食细菌给线虫的方式让线虫得以产生RNA干扰现象。RNAi现象在生物中普遍存在。2006年，安德鲁·法厄（Andrew Z. Fire）与克雷格·梅洛（Craig C. Mello）由于在1998年发表对于秀丽隐杆线虫的RNAi机制研究中的贡献而共同获得诺贝尔生理及医学奖。

RNAi在不同文献上有不同的称呼，如转录后基因沉默（post-transcriptional gene silencing and transgene silencing, PTGS）、共同抑制（co-suppression），皆是指RNA干扰。

RNA干扰中有两种小RNA扮演了重要的角色，分别是微RNA（miRNA）和小干扰RNA（siRNA）。这些RNA会结合到特定的mRNA，调控其活性，借此可以直接调控蛋白质的制造。RNAi在细胞防御上扮演重要的角色，可以降解外来的核酸序列，如病毒及转位子。另外也与发育有关。

许多真核生物可以找到RNAi的现象。其起始机制是由裁切子（英语：Dicer）（Dicer）负责，它是一种可将长链双股RNA（dsRNA）的酵素切为20个核苷酸以下的siRNA。每一段siRNA会被拆开为两个单股RNA（ssRNA），分别为称为随从股（passenger strand）和向导股（guide strand）。随从股会被降解掉，而向导股则会与RNA诱导沉默复合体（RISC）结合。与RISC结合的向导股会找到相对应的mRNA序列，这时候RISC的酵素活性单元Argonaute会将此mRNA降解。在某些生物，这个反应会系统性地增强，以极少量的siRNA达到相当显著的转译抑制效果。

RNAi不论是在细胞培养还是活体动物内都是一项有力的研究工具，因为只要将人工合成的dsRNA送入细胞内，便可精准的抑制特定基因表现，据此可以判断某基因在细胞内所扮演的角色。这门技术广泛应用于生物科技、医学，以及杀虫剂研究上。[4]

细胞机制

梨形鞭毛虫体内的裁切子（英语：Dicer）（Dicer）蛋白可以将dsRNA切为siRNA。绿色的结构域为RNA酶，黄色的为PAZ结构域，红色的为platform结构域，蓝色的为连接螺旋（connector helix）。[5]

RNA干扰属于一种RNA依赖性基因沉默作用，负责这个作用起始部分的是一种名为RNA诱导沉默复合体（RISC）的蛋白。一般来说，细胞质内的RNA通常为单股。一旦出现双股RNA（dsRNA），即会立即被裁切子（Dicer）切为短片段。此时，RISC中的活性单位argonaute部分便会与之作用，引起RNA干扰。[6]

可引起RNA干扰的dsRNA可分为外源性（细胞外侵入）和内源性（细胞内自行产生）。外源性dsRNA可能来自于病毒感染或实验室胞内注射；内源性则来自于RNA编码基因转录的前微小RNA。前微小RNA会在细胞核内形成特有的茎环结构，之后再送入细胞质内。[6][7]

dsRNA剪切

内源性的双股RNA（dsRNA）会刺激一种叫做裁切子（英语：dicer）的RNA酶，启动RNA干扰程序[8]。裁切子会先将dsRNA从3'端突出的两个核苷酸，逐步切成20至25碱基对长的短片段[9]。 经过生物信息学分析大量生物的基因体后，发现20至25碱基对长的片段，可使目标基因的专一性最大化，也能够减少其他非特定的副作用。[10]被裁切子切下来的短片段称为小干扰RNA（siRNA）。siRNA会分为两股并与RISC结合，一旦侦测到有与siRNA相合的mRNA，RISC中的酵素活性区域Argonaute会将其降解，阻断之后的转译作用。[11]

外源性dsRNA则是由一种效应蛋白侦测并结合，如秀丽隐杆线虫身上的RDE-4，和果蝇的R2D2。这些效应蛋白可以刺激裁切子的活性[12]。这类效应蛋白只会与长双股RNA结合，但对于长度专一性的原因至今仍不清楚[12]这种RNA结合蛋白会促使已经被切下来的siRNA与RISC结合。[13]

秀丽隐杆线虫会借由RNA依赖性RNA聚合酶（RdRP）合成大量的“次级”siRNA来放大RNAi的效果[14][15]这些“次级”siRNA会在结构上与被裁切子切下来的siRNA相当不同[15][16]

微小RNA

主条目：微小RNA
甘蓝前微小RNA的二级结构，可以看见当中具有茎环

微小RNA（miRNAs）属于一种非编码RNA，负责调控基因表现，特别是在发育上[17]。成熟的miRNA需经过一连串转录后修饰，在结构上与siRNA相似，只是miRNA是细胞内自行产生，siRNA则是来自于外来的双股RNA[18]。

刚从DNA初步转录下来的miRNA前驱物称为“初级微小RNA”（pri-miRNA），位于细胞核中。之后微处理器复合体（microprocessor complex）会将初级微小RNA修饰微“前微小RNA”（pre-miRNA），产生一个70核苷酸大小的茎环。微处理器复合体含有一种称为Drosha的RNA酶III，以及一种双股RNA结合蛋白DGCR8。此时裁切子会将前微小RNA的双股部分切下，切下的片段就是成熟的miRNA，可与RISC结合。自此，miRNA及siRNA皆会以相同的步骤，完成RNA干扰的程序[18]。


siRNA与miRNA的对于目标基因的相合度，特别是在动物身上。miRNA对于目标基因通常不会完全相合，相对的siRNA通常对于目标基因可以完美地相合[19]。在黑腹果蝇和秀丽隐杆线虫身上，miRNA和siRNA是分别由特定的argonaute蛋白和裁切子处理[20][21]。

RISC的活化与催化反应

左：从古菌激烈火球菌（英语：Pyrococcus furiosus）体内取得的全argonaute蛋白。
右：argonaute的PIWI结构域（英语：PIWI domain）与dsRNA所形成的复合物

RISC是由许多种酵素所结合而成的大型复合体，其中具有内切酶活性的部分称为Argonaute。这种内切酶可以降解与siRNA模版互补的RNA[6]。裁切子切下的RNA片段皆为双股，照理来说，应该两股都能够形成siRNA，然而却只有一股能够与argonaute结合进行RNA干扰作用，另一股则会再结合过程中被RISC降解。我们称能够与RISC结合的一股为“向导股”（guide strand），另一股则称为“反向导股”（anti-guide strand）或“随从股”（passenger strand）[22]。

一开始，学界以为分开两股的是一种ATP依赖螺旋酶[23]。但后来研究才发现RISC的酶活性单元会直接解开两股[24][25]。然而，RNA干扰的活体外动力学分析，发现ATP可能对于将剪切完后的mRNA从RISC移除的过程中，扮演重要角色[26]。向导股有一个特色，即其5'端对于其互补序列结合性较弱[27]，但如何选择向导股仍不清楚。目前已知其选择性与siRNA以及RISC结合的倾向无关[注 1][28]。相对的，R2D2蛋白则可能扮演稳定随从股5'端的角色。[29]

RNA与RISC结合的结构已经以X射线晶体学探知，与RNA结合的结构域为argonaute蛋白。 Here, the phosphorylated 5' end of the RNA strand enters a conserved basic surface pocket and makes contacts through a divalent cation (an atom with two positive charges) such as magnesium and by aromatic stacking (a process that allows more than one atom to share an electron by passing it back and forth) between the 5' nucleotide in the siRNA and a conserved tyrosine residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target.[30] Analysis of the inhibitory effect of mismatches in either the 5’ or 3’ end of the guide strand has demonstrated that the 5’ end of the guide strand is likely responsible for matching and binding the target mRNA, while the 3’ end is responsible for physically arranging target mRNA into a cleavage-favorable RISC region.[26]

It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation, translation of the mRNA target is not essential for RNAi-mediated degradation.[31] Indeed, RNAi may be more effective against mRNA targets that are not translated.[32] Argonaute proteins are localized to specific regions in the cytoplasm called P-bodies (also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay;[33] miRNA activity is also clustered in P-bodies.[34] Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are a critical site in the RNAi process.[35]

Transcriptional silencing

The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA. These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation.[36]

Components of the RNAi pathway are used in many eukaryotes in the maintenance of the organization and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serves to downregulate genes pre-transcriptionally;[37] this process is referred to as RNA-induced transcriptional silencing (RITS), and is carried out by a complex of proteins called the RITS complex. In fission yeast this complex contains argonaute, a chromodomain protein Chp1, and a protein called Tas3 of unknown function.[38] As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins.[39] Indeed, deletion of these genes in the fission yeast S. pombe disrupts histone methylation and centromere formation,[40] causing slow or stalled anaphase during cell division.[41] In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.[42]

RNA干扰现象的机理[43]

The mechanism by which the RITS complex induces heterochromatin formation and organization is not well understood. Most studies have focused on the mating-type region in fission yeast, which may not be representative of activities in other genomic regions/organisms. In maintenance of existing heterochromatin regions, RITS forms a complex with siRNAs complementary to the local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that are initiated by RNA polymerase. The formation of such a heterochromatin region, though not its maintenance, is dicer-dependent, presumably because dicer is required to generate the initial complement of siRNAs that target subsequent transcripts.[44] Heterochromatin maintenance has been suggested to function as a self-reinforcing feedback loop, as new siRNAs are formed from the occasional nascent transcripts by RdRP for incorporation into local RITS complexes.[45] The relevance of observations from fission yeast mating-type regions and centromeres to mammals is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.[46]

Crosstalk with RNA editing

The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine nucleotides into inosine in dsRNAs via the enzyme adenosine deaminase (ADAR).[47] It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate.[48] Some pre-miRNAs do undergo A→I RNA editing[49][50] and this mechanism may regulate the processing and expression of mature miRNAs.[50] Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components.[51] Further support for this model comes from studies on ADAR-null C. elegans strains indicating that A→I RNA editing may counteract RNAi silencing of endogenous genes and transgenes.[52]

Illustration of the major differences between plant and animal gene silencing. Natively expressed microRNA or exogenous small interfering RNA is processed by dicer and integrated into the RISC complex, which mediates gene silencing.[53]

Variation among organisms

Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and C. elegans, although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata (channels in the cell walls that enable communication and transport).[23] Heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell.[54] A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression.[53] This translational effect may be produced by inhibiting the interactions of translation initiation factors with the messenger RNA's polyadenine tail.[55]

Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway entirely.[56][57] Most or all of the components are also missing in some fungi, most notably the model organism Saccharomyces cerevisiae.[58] The presence of RNAi in other budding yeast species such as Saccharomyces castellii and Candida albicans, further demonstrates that inducing two RNAi-related proteins from S. castellii facilitates RNAi in S. cerevisiae.[59] That certain ascomycetes and basidiomycetes are missing RNA interference pathways indicates that proteins required for RNA silencing have been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niches.[60]

Gene expression in prokaryotes is influenced by an RNA-based system similar in some respects to RNAi. Here, RNA-encoding genes control mRNA abundance or translation by producing a complementary RNA that anneals to an mRNA. However these regulatory RNAs are not generally considered to be analogous to miRNAs because the dicer enzyme is not involved.[61] It has been suggested that CRISPR interference systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components are orthologous.[62]



RNA干扰的作用

2001年，Tuschl等将siRNA导入到哺乳动物细胞中并由此解决了在哺乳细胞内导入长的双链RNA时引发的干扰素效应，由此拓展了RNAi在基因治疗上应用前景[来源请求]。RNAi机制普遍存在于动植物，尤其是低等生物中[63]。因此被认为是进化上相对保守的基因表达调控机制[来源请求]。一种假说为[63]，RNAi机制是作为在RNA水平上抵御病毒入侵的防御机制而存在的。在病毒自身基因组所包含的，或在病毒复制过程中产生的双链RNA可以被Dicer识别，从而引起病毒RNA降解。但是许多病毒为抵抗宿主的RNA干扰机制，会产生抑制宿主RNA干扰的蛋白，以保护病毒基因在宿主体内的顺利复制。已经发现的可以抑制宿主RNA干扰的病毒蛋白有potyviruses编码的HC-PRO蛋白、马铃薯X病毒编码的Cmv2b蛋白、兽棚病毒编码的B2蛋白等[64]。

RNA干扰也是抑制破坏基因结构的一种DNA片段转录子活性的重要方式。转录子通常以逆转录的方式在基因组中扩增。在逆转录过程中产生的双链RNA分子可以被Dicer识别，从而被降解[63]。

目前发现[来源请求]，RNAi机制中的相关一些因子如内源性双链RNA及蛋白因子可以在多种层次上对基因表达进行调控，其范围已经超越了PTGS（post transcriptional gene silencing），如RNAi机制同样参与了转录水平上的基因表达调控过程中。

Biological functions

Immunity

RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may also prevent the self-propagation of transposons.[65] Plants such as Arabidopsis thaliana express multiple dicer homologs that are specialized to react differently when the plant is exposed to different viruses.[66] Even before the RNAi pathway was fully understood, it was known that induced gene silencing in plants could spread throughout the plant in a systemic effect and could be transferred from stock to scion plants via grafting.[67] This phenomenon has since been recognized as a feature of the plant adaptive immune system and allows the entire plant to respond to a virus after an initial localized encounter.[68] In response, many plant viruses have evolved elaborate mechanisms to suppress the RNAi response.[69] These include viral proteins that bind short double-stranded RNA fragments with single-stranded overhang ends, such as those produced by dicer.[70] Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria.[71] These effects may be part of a generalized response to pathogens that downregulates any metabolic process in the host that aids the infection process.[72]

Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals produces an antiviral response. In both juvenile and adult Drosophila, RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus.[73][74] A similar role in immunity may operate in C. elegans, as argonaute proteins are upregulated in response to viruses and worms that overexpress components of the RNAi pathway are resistant to viral infection.[75][76]

The role of RNA interference in mammalian innate immunity is poorly understood, and relatively little data is available. However, the existence of viruses that encode genes able to suppress the RNAi response in mammalian cells may be evidence in favour of an RNAi-dependent mammalian immune response,[77][78] although this hypothesis has been challenged as poorly substantiated.[79] Maillard et al.[80] and Li et al.[81] provide evidence for the existence of a functional antiviral RNAi pathway in mammalian cells. Other functions for RNAi in mammalian viruses also exist, such as miRNAs expressed by the herpes virus that may act as heterochromatin organization triggers to mediate viral latency.[42]

Downregulation of genes

Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most important in translational repression[53] and in the regulation of development, especially on the timing of morphogenesis and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cells.[82] The role of endogenously expressed miRNA in downregulating gene expression was first described in C. elegans in 1993.[83] In plants this function was discovered when the "JAW microRNA" of Arabidopsis was shown to be involved in the regulation of several genes that control plant shape.[84] In plants, the majority of genes regulated by miRNAs are transcription factors;[85] thus miRNA activity is particularly wide-ranging and regulates entire gene networks during development by modulating the expression of key regulatory genes, including transcription factors as well as F-box proteins.[86] In many organisms, including humans, miRNAs are linked to the formation of tumors and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumor suppressors.[87]

Upregulation of genes

RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon dubbed RNA activation. Part of the mechanism for how these RNA upregulate genes is known: dicer and argonaute are involved, possibly via histone demethylation.[88] miRNAs have been proposed to upregulate their target genes upon cell cycle arrest, via unknown mechanisms.[89]

Evolution

Based on parsimony-based phylogenetic analysis, the most recent common ancestor of all eukaryotes most likely already possessed an early RNA interference pathway; the absence of the pathway in certain eukaryotes is thought to be a derived characteristic.[90] This ancestral RNAi system probably contained at least one dicer-like protein, one argonaute, one PIWI protein, and an RNA-dependent RNA polymerase that may also have played other cellular roles. A large-scale comparative genomics study likewise indicates that the eukaryotic crown group already possessed these components, which may then have had closer functional associations with generalized RNA degradation systems such as the exosome.[91] This study also suggests that the RNA-binding argonaute protein family, which is shared among eukaryotes, most archaea, and at least some bacteria (such as Aquifex aeolicus), is homologous to and originally evolved from components of the translation initiation system.

The ancestral function of the RNAi system is generally agreed to have been immune defense against exogenous genetic elements such as transposons and viral genomes.[90][92] Related functions such as histone modification may have already been present in the ancestor of modern eukaryotes, although other functions such as regulation of development by miRNA are thought to have evolved later.[90]

RNA interference genes, as components of the antiviral innate immune system in many eukaryotes, are involved in an evolutionary arms race with viral genes. Some viruses have evolved mechanisms for suppressing the RNAi response in their host cells, particularly for plant viruses.[69] Studies of evolutionary rates in Drosophila have shown that genes in the RNAi pathway are subject to strong directional selection and are among the fastest-evolving genes in the Drosophila genome.[93]

Applications

Gene knockdown

The RNA interference pathway is often exploited in experimental biology to study the function of genes in cell culture and in vivo in model organisms.[6] Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated.[94]

Extensive efforts in computational biology have been directed toward the design of successful dsRNA reagents that maximize gene knockdown but minimize "off-target" effects. Off-target effects arise when an introduced RNA has a base sequence that can pair with and thus reduce the expression of multiple genes. Such problems occur more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of humans, C. elegans and S. pombe that about 10% of possible siRNAs have substantial off-target effects.[10] A multitude of software tools have been developed implementing algorithms for the design of general[95][96] mammal-specific,[97] and virus-specific[98] siRNAs that are automatically checked for possible cross-reactivity.

Depending on the organism and experimental system, the exogenous RNA may be a long strand designed to be cleaved by dicer, or short RNAs designed to serve as siRNA substrates. In most mammalian cells, shorter RNAs are used because long double-stranded RNA molecules induce the mammalian interferon response, a form of innate immunity that reacts nonspecifically to foreign genetic material.[99] Mouse oocytes and cells from early mouse embryos lack this reaction to exogenous dsRNA and are therefore a common model system for studying mammalian gene-knockdown effects.[100] Specialized laboratory techniques have also been developed to improve the utility of RNAi in mammalian systems by avoiding the direct introduction of siRNA, for example, by stable transfection with a plasmid encoding the appropriate sequence from which siRNAs can be transcribed,[101] or by more elaborate lentiviral vector systems allowing the inducible activation or deactivation of transcription, known as conditional RNAi.[102][103]

Functional genomics

A normal adult Drosophila fly, a common model organism used in RNAi experiments.

Most functional genomics applications of RNAi in animals have used C. elegans[104] and Drosophila,[105] as these are the common model organisms in which RNAi is most effective. C. elegans is particularly useful for RNAi research for two reasons: firstly, the effects of gene silencing are generally heritable, and secondly because delivery of the dsRNA is extremely simple. Through a mechanism whose details are poorly understood, bacteria such as E. coli that carry the desired dsRNA can be fed to the worms and will transfer their RNA payload to the worm via the intestinal tract. This "delivery by feeding" is just as effective at inducing gene silencing as more costly and time-consuming delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads.[106] Although delivery is more difficult in most other organisms, efforts are also underway to undertake large-scale genomic screening applications in cell culture with mammalian cells.[107]

Approaches to the design of genome-wide RNAi libraries can require more sophistication than the design of a single siRNA for a defined set of experimental conditions. Artificial neural networks are frequently used to design siRNA libraries[108] and to predict their likely efficiency at gene knockdown.[109] Mass genomic screening is widely seen as a promising method for genome annotation and has triggered the development of high-throughput screening methods based on microarrays.[110][111] However, the utility of these screens and the ability of techniques developed on model organisms to generalize to even closely related species has been questioned, for example from C. elegans to related parasitic nematodes.[112][113]

Functional genomics using RNAi is a particularly attractive technique for genomic mapping and annotation in plants because many plants are polyploid, which presents substantial challenges for more traditional genetic engineering methods. For example, RNAi has been successfully used for functional genomics studies in bread wheat (which is hexaploid)[114] as well as more common plant model systems Arabidopsis and maize.[115]

Medicine

参见：Therapeutic gene modulation
An adult C. elegans worm, grown under RNAi suppression of a nuclear hormone receptor involved in desaturase regulation. These worms have abnormal fatty acid metabolism but are viable and fertile.[116]

It may be possible to exploit RNA interference in therapy. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA has been more successful.[117] Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus.[118] RNAi has also been shown to be effective in reversing induced liver failure in mouse models.[119]

Antiviral

Potential antiviral therapies include topical microbicide treatments that use RNAi to treat infection (at Harvard Medical School; in mice, so far) by herpes simplex virus type 2 and the inhibition of viral gene expression in cancerous cells,[120] knockdown of host receptors and coreceptors for HIV,[121] the silencing of hepatitis A[122] and hepatitis B genes,[123] silencing of influenza gene expression,[42] and inhibition of measles viral replication.[124] Potential treatments for neurodegenerative diseases have also been proposed, with particular attention to polyglutamine diseases such as Huntington's disease.[125]

RNA interference-based applications are being developed to target persistent HIV-1 infection. Viruses like HIV-1 are particularly difficult targets for RNAi-attack because they are escape-prone, which requires combinatorial RNAi strategies to prevent viral escape.[126]

Cancer

RNA interference is also a promising way to treat cancers by silencing genes differentially upregulated in tumor cells or genes involved in cell division.[127][128] A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy.[129][130]

Due to safety concerns with viral vectors, nonviral delivery methods, typically employing lipid-based[131] or polymeric[132] vectors, are also promising candidates. Computational modeling of nonviral siRNA delivery paired with in vitro and in vivo gene knockdown studies elucidated the temporal behavior of RNAi in these systems. The model used an input bolus dose of siRNA and computationally and experimentally showed that knockdown duration was dependent mainly on the doubling time of the cells to which siRNA was delivered, while peak knockdown depended primarily on the delivered dose. Kinetic considerations of RNAi are imperative to safe and effective dosing schedules as nonviral methods of inducing RNAi continue to be developed.[133][134]

Safety

Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed.[135] A computational genomics study estimated that the error rate of off-target interactions is about 10%.[10] One major study of liver disease in mice reported that 23 out of 49 distinct RNAi treatment protocols resulted in death.[136] Researchers hypothesized this alarmingly high rate to be the result of "oversaturation" of the dsRNA pathway,[137] due to the use of shRNAs that have to be processed in the nucleus and exported to the cytoplasm using an active mechanism. Such considerations are under active investigation, to reduce their impact in the potential therapeutic applications.

RNAi in vivo delivery to tissues still eludes science—especially to tissues deep within the body. RNAi delivery is only easily accessible to surface tissues such as the eye and respiratory tract. In these instances, siRNA has been used in direct contact with the tissue for transport. The resulting RNAi successfully focused on target genes. When delivering siRNA to deep tissues, the siRNA must be protected from nucleases, but targeting specific areas becomes the main difficulty. This difficulty has been combatted with high dosage levels of siRNA to ensure the tissues have been reached, however in these cases hepatotoxicity was reported.[137]

Biotechnology

RNA interference has been used for applications in biotechnology and is nearing commercialization in others. RNAi has developed many novel crops such as nicotinefree tobacco, decaffeinated coffee, nutrient fortified and hypoallergenic crops. The genetically engineered Arctic apples are near close to receive US approval. The apples were produced by RNAi suppression of PPO (polyphenol oxidase) gene making apple varieties that will not undergo browning after being sliced. PPO-silenced apples are unable to convert chlorogenic acid into quinone product.[138]

There are several opportunities for the applications of RNAi in crop science for its improvement such as stress tolerance and enhanced nutritional level. RNAi will prove its potential for inhibition of photorespiration to enhance the productivity of C3 plants. This knockdown technology may be useful in inducing early flowering, delayed ripening, delayed senescence, breaking dormancy, stress-free plants, overcoming self-sterility, etc.[138]

Foods

RNAi has been used to genetically engineer plants to produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. Cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is itself important in preventing damage from plant pests.[139] Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.[140]

No plant products that use RNAi-based genetic engineering have yet exited the experimental stage. Development efforts have successfully reduced the levels of allergens in tomato plants[141] and fortification of plants such as tomatoes with dietary antioxidants.[142] Previous commercial products, including the Flavr Savr tomato and two cultivars of ringspot-resistant papaya, were originally developed using antisense technology but likely exploited the RNAi pathway.[143][144]

Other crops

Another effort decreased the precursors of likely carcinogens in tobacco plants.[145] Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy[146] and resistance to common plant viruses.[147]

Insecticide

RNAi is under development as an insecticide, employing multiple approaches, including genetic engineering and topical application. Cells in the midgut of many larvae take up the molecules and help spread the signal throughout the insect's body.[4]

RNAi has varying effects in different species of Lepidoptera (butterflies and moths).[148] Possibly because their saliva is better at breaking down RNA, the cotton bollworm, the beet armyworm and the Asiatic rice borer have so far not been proven susceptible to RNAi by feeding.[4]

To develop resistance to RNAi, the western corn rootworm would have to change the genetic sequence of its Snf7 gene at multiple sites. Combining multiple strategies, such as engineering the protein Cry, derived from a bacterium called Bacillus thuringiensis (Bt), and RNAi in one plant delay the onset of resistance.[4]

One unconfirmed 2012 paper detected small RNAs from food plants in the blood of mice and humans. The consequences of RNA insecticides in the human bloodstream have not been investigated. Biological barriers—including saliva and blood enzymes and stomach acids may break down any ingested RNA. Critics charge that the human equivalent of the mouse diet in the study would be 33 kilograms of cooked rice a day. Two 2013 studies failed to detect RNAs in humans. Athletes consuming a diet of apples and bananas and monkeys consuming a fruit shake both appeared to be RNA-free.[4]

Transgenic plants

Transgenic crops have been made to express small bits of RNA, carefully chosen to silence crucial genes in target pests. RNAs exist that affect only insects that have specific genetic sequences. In 2009 a study showed RNAs that could kill any one of four fruit fly species while not harming the other three.[4]

In 2012 Syngenta bought Belgian RNAi firm Devgen for $522 million and Monsanto paid $29.2 million for the exclusive rights to intellectual property from Alnylam Pharmaceuticals. The International Potato Center in Lima, Peru is looking for genes to target in the sweet potato weevil, a beetle whose larvae ravage sweet potatoes globally. Other researchers are trying to silence genes in ants, caterpillars and pollen beetles. Monsanto will likely be first to market, with a transgenic corn seed that expresses dsRNA based on gene Snf7 from the western corn rootworm, a beetle whose larvae annually cause one billion dollars in damage in the United States alone. A 2012 paper showed that silencing Snf7 stunts larval growth, killing them within days. In 2013 the same team showed that the RNA affects very few other species.[4]

Topical

Alternatively dsRNA can be supplied without genetic engineering. One approach is to add them to irrigation water. The molecules are absorbed into the plants' vascular system and poison insects feeding on them. Another approach involves spraying RNA like a conventional pesticide. This would allow faster adaptation to resistance. Such approaches would require low cost sources of RNAs that do not currently exist.[4]

Genome-scale screening

Genome-scale RNAi research relies on high-throughput screening (HTS) technology. RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific phenotypes. This technology has been hailed as the second genomics wave, following the first genomics wave of gene expression microarray and single nucleotide polymorphism discovery platforms.[149] One major advantage of genome-scale RNAi screening is its ability to simultaneously interrogate thousands of genes. With the ability to generate a large amount of data per experiment, genome-scale RNAi screening has led to an explosion data generation rates. Exploiting such large data sets is a fundamental challenge, requiring suitable statistics/bioinformatics methods. The basic process of cell-based RNAi screening includes the choice of an RNAi library, robust and stable cell types, transfection with RNAi agents, treatment/incubation, signal detection, analysis and identification of important genes or therapeutical targets.[150]

历史

以RNAi技术抑制矮牵牛花的色素基因。左边的为野生种，右边的为利用转位子破坏色素基因的结果，中间的则为RNAi操作结果[151]

RNA干扰最早是在植物体身上发现[152]。1990年由约根森（Jorgensen）研究小组为得到颜色更深的矮牵牛花，而过量表达查尔酮合成酶（英语：chalcone synthase），以得到更多的粉红色及紫色色素。结果意外得到了白色和白紫杂色的矮牵牛花，并且过量表达查尔酮合成酶的矮牵牛花中查尔酮合成酶的浓度比正常矮牵牛花中的浓度低50倍。约根森推测外源转入的编码查尔酮合成酶的基因同时抑制了花中内源查尔酮合成酶基因的表达[153]。后来研究者又在粉色面包霉菌上发现了类似的现象[154]，但当时没有人意会到两者是同一现象。进一步研究发现，这些基因抑制现象都源自于mRNA的降解[155]，并称此现象为“基因表现的共同抑制”（co-suppression of gene expression），但此时详细机制并不清楚[156]。

不久之后，植物病毒学家在一个希望改良植物对于病毒抵抗力的实验中，发现了类似的现象。当时已知植物会表现抗病毒蛋白，加强自身抵抗力。但并不知道这些病毒带来的RNA短片段，居然也能够使植物产生免疫力。因此，研究者推测将病毒基因插入植物基因体中应该也可以得到相同的免疫力[157]。同时，研究者也将植物基因送入病毒，使病毒感植物体。结果植物的基因被抑制，此现象称为“病毒诱导基因沉默”（virus-induced gene silencing，VIGS）。而这一类类似的现象则统称为“转录后基因沉默”（post transcriptional gene silencing）[158]。

After these initial observations in plants, laboratories searched for this phenomenon in other organisms.[159][160] Craig C. Mello and Andrew Fire's 1998 Nature paper reported a potent gene silencing effect after injecting double stranded RNA into C. elegans.[1] In investigating the regulation of muscle protein production, they observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term RNAi. This discovery represented the first identification of the causative agent for the phenomenon. Fire and Mello were awarded the 2006 Nobel Prize in Physiology or Medicine.[6]


1992年，罗马诺（Romano）和Macino也在粗糙链孢霉中发现了外源导入基因可以抑制具有同源序列的内源基因的表达[161]。1995年，Guo和Kemphues在线虫中也发现了RNA干扰现象[162]。

1998年，安德鲁·法厄（Andrew Z. Fire）等在秀丽隐杆线虫（C.elegans）中进行反义RNA抑制实验时发现，作为对照加入的双链RNA相比正义或反义RNA显示出了更强的抑制效果[1]。从与靶mRNA的分子量比考虑，加入的双链RNA的抑制效果要强于理论上1:1配对时的抑制效果，因此推测在双链RNA引导的抑制过程中存在某种扩增效应并且有某种酶活性参与其中。并且将这种现象命名为RNA干扰。

2006年，安德鲁·法厄与克雷格·梅洛（Craig C. Mello）由于在RNAi机制研究中的贡献获得诺贝尔生理及医学奖。

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书籍

  • 《新药药物靶标开发技术》，2006年版，高等教育出版社，ISBN 7-04-018953-4

期刊文章

  • Recent develpment of RNAi in drug target discovery and validation, Drug Disvoery Today:Technologies.(2006)3:293-300.
  • Development of new RNAi therapeutics, Histology and Histopathology. (2007)22:211-217.


外部链接

维基共享资源上的相关多媒体资源：Koala0090/sandbox6
维基学院中的相关研究或学习资源：Koala0090/sandbox6

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