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慢病毒會設計shRNA並注入到哺乳動物細胞中，進行RNA干擾。
RNAi示意圖

RNA干擾（RNA interference，縮寫為RNAi）是指一種分子生物學上由雙鏈RNA誘發的基因沉默現象，其機制是通過阻礙特定基因的轉譯或轉錄來抑制基因表達。當細胞中導入與內源性mRNA編碼區同源的雙鏈RNA時，該mRNA發生降解而導致基因表達沉默[1]。與其它基因沉默現象不同的是，在植物和線蟲中，RNAi具有傳遞性，可在細胞之間傳播，此現象被稱作系統性RNA干擾（systemic RNAi）[2][3]。在秀麗隱杆線蟲上實驗時還可使子一代產生基因突變，甚至於可用餵食細菌給線蟲的方式讓線蟲得以產生RNA干擾現象。RNAi現象在生物中普遍存在。2006年，安德魯·法厄（Andrew Z. Fire）與克雷格·梅洛（Craig C. Mello）由於在1998年發表對於秀麗隱杆線蟲的RNAi機制研究中的貢獻而共同獲得諾貝爾生理及醫學獎。

RNAi在不同文獻上有不同的稱呼，如轉錄後基因沉默（post-transcriptional gene silencing and transgene silencing, PTGS）、共同抑制（co-suppression），皆是指RNA干擾。

RNA干擾中有兩種小RNA扮演了重要的角色，分別是微RNA（miRNA）和小干擾RNA（siRNA）。這些RNA會結合到特定的mRNA，調控其活性，藉此可以直接調控蛋白質的製造。RNAi在細胞防禦上扮演重要的角色，可以降解外來的核酸序列，如病毒及轉位子。另外也與發育有關。

許多真核生物可以找到RNAi的現象。其起始機制是由裁切子（英語：Dicer）（Dicer）負責，它是一種可將長鏈雙股RNA（dsRNA）的酵素切為20個核苷酸以下的siRNA。每一段siRNA會被拆開為兩個單股RNA（ssRNA），分別為稱為隨從股（passenger strand）和嚮導股（guide strand）。隨從股會被降解掉，而嚮導股則會與RNA誘導沉默複合體（RISC）結合。與RISC結合的嚮導股會找到相對應的mRNA序列，這時候RISC的酵素活性單元Argonaute會將此mRNA降解。在某些生物，這個反應會系統性地增強，以極少量的siRNA達到相當顯著的轉譯抑制效果。

RNAi不論是在細胞培養還是活體動物內都是一項有力的研究工具，因為只要將人工合成的dsRNA送入細胞內，便可精準的抑制特定基因表現，據此可以判斷某基因在細胞內所扮演的角色。這門技術廣泛應用於生物科技、醫學，以及殺蟲劑研究上。[4]

細胞機制

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梨形鞭毛蟲體內的裁切子（英語：Dicer）（Dicer）蛋白可以將dsRNA切為siRNA。綠色的結構域為RNA酶，黃色的為PAZ結構域，紅色的為platform結構域，藍色的為連接螺旋（connector helix）。[5]

RNA干擾屬於一種RNA依賴性基因沉默作用，負責這個作用起始部分的是一種名為RNA誘導沉默複合體（RISC）的蛋白。一般來說，細胞質內的RNA通常為單股。一旦出現雙股RNA（dsRNA），即會立即被裁切子（Dicer）切為短片段。此時，RISC中的活性單位argonaute部分便會與之作用，引起RNA干擾。[6]

可引起RNA干擾的dsRNA可分為外源性（細胞外侵入）和內源性（細胞內自行產生）。外源性dsRNA可能來自於病毒感染或實驗室胞內注射；內源性則來自於RNA編碼基因轉錄的前微小RNA。前微小RNA會在細胞核內形成特有的莖環結構，之後再送入細胞質內。[6][7]

dsRNA剪切

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內源性的雙股RNA（dsRNA）會刺激一種叫做裁切子（英語：dicer）的RNA酶，啟動RNA干擾程序[8]。裁切子會先將dsRNA從3'端突出的兩個核苷酸，逐步切成20至25鹼基對長的短片段[9]。 經過生物信息學分析大量生物的基因體後，發現20至25鹼基對長的片段，可使目標基因的專一性最大化，也能夠減少其他非特定的副作用。[10]被裁切子切下來的短片段稱為小干擾RNA（siRNA）。siRNA會分為兩股並與RISC結合，一旦偵測到有與siRNA相合的mRNA，RISC中的酵素活性區域Argonaute會將其降解，阻斷之後的轉譯作用。[11]

外源性dsRNA則是由一種效應蛋白偵測並結合，如秀麗隱桿線蟲身上的RDE-4，和果蠅的R2D2。這些效應蛋白可以刺激裁切子的活性[12]。這類效應蛋白只會與長雙股RNA結合，但對於長度專一性的原因至今仍不清楚[12]這種RNA結合蛋白會促使已經被切下來的siRNA與RISC結合。[13]

秀麗隱桿線蟲會藉由RNA依賴性RNA聚合酶（RdRP）合成大量的「次級」siRNA來放大RNAi的效果[14][15]這些「次級」siRNA會在結構上與被裁切子切下來的siRNA相當不同[15][16]

微小RNA

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主條目：微小RNA
 
甘藍前微小RNA的二級結構，可以看見當中具有莖環

微小RNA（miRNAs）屬於一種非編碼RNA，負責調控基因表現，特別是在發育上[17]。成熟的miRNA需經過一連串轉錄後修飾，在結構上與siRNA相似，只是miRNA是細胞內自行產生，siRNA則是來自於外來的雙股RNA[18]。

剛從DNA初步轉錄下來的miRNA前驅物稱為「初級微小RNA」（pri-miRNA），位於細胞核中。之後微處理器複合體（microprocessor complex）會將初級微小RNA修飾微「前微小RNA」（pre-miRNA），產生一個70核苷酸大小的莖環。微處理器複合體含有一種稱為Drosha的RNA酶III，以及一種雙股RNA結合蛋白DGCR8。此時裁切子會將前微小RNA的雙股部分切下，切下的片段就是成熟的miRNA，可與RISC結合。自此，miRNA及siRNA皆會以相同的步驟，完成RNA干擾的程序[18]。


siRNA與miRNA的對於目標基因的相合度，特別是在動物身上。miRNA對於目標基因通常不會完全相合，相對的siRNA通常對於目標基因可以完美地相合[19]。在黑腹果蠅和秀麗隱桿線蟲身上，miRNA和siRNA是分別由特定的argonaute蛋白和裁切子處理[20][21]。

RISC的活化與催化反應

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左：從古菌激烈火球菌（英語：Pyrococcus furiosus）體內取得的全argonaute蛋白。
右：argonaute的PIWI結構域（英語：PIWI domain）與dsRNA所形成的複合物

RISC是由許多種酵素所結合而成的大型複合體，其中具有內切酶活性的部分稱為Argonaute。這種內切酶可以降解與siRNA模版互補的RNA[6]。裁切子切下的RNA片段皆為雙股，照理來說，應該兩股都能夠形成siRNA，然而卻只有一股能夠與argonaute結合進行RNA干擾作用，另一股則會再結合過程中被RISC降解。我們稱能夠與RISC結合的一股為「嚮導股」（guide strand），另一股則稱為「反嚮導股」（anti-guide strand）或「隨從股」（passenger strand）[22]。

一開始，學界以為分開兩股的是一種ATP依賴螺旋酶[23]。但後來研究才發現RISC的酶活性單元會直接解開兩股[24][25]。然而，RNA干擾的活體外動力學分析，發現ATP可能對於將剪切完後的mRNA從RISC移除的過程中，扮演重要角色[26]。嚮導股有一個特色，即其5'端對於其互補序列結合性較弱[27]，但如何選擇嚮導股仍不清楚。目前已知其選擇性與siRNA以及RISC結合的傾向無關[註 1][28]。相對的，R2D2蛋白則可能扮演穩定隨從股5'端的角色。[29]

RNA與RISC結合的結構已經以X射線晶體學探知，與RNA結合的結構域為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

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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干擾現象的機理[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

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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

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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]

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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干擾的作用

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2001年，Tuschl等將siRNA導入到哺乳動物細胞中並由此解決了在哺乳細胞內導入長的雙鏈RNA時引發的干擾素效應，由此拓展了RNAi在基因治療上應用前景[來源請求]。RNAi機制普遍存在於動植物，尤其是低等生物中[63]。因此被認為是進化上相對保守的基因表達調控機制[來源請求]。一種假說為[63]，RNAi機制是作為在RNA水平上抵禦病毒入侵的防禦機制而存在的。在病毒自身基因組所包含的，或在病毒複製過程中產生的雙鏈RNA可以被Dicer識別，從而引起病毒RNA降解。但是許多病毒為抵抗宿主的RNA干擾機制，會產生抑制宿主RNA干擾的蛋白，以保護病毒基因在宿主體內的順利複製。已經發現的可以抑制宿主RNA干擾的病毒蛋白有potyviruses編碼的HC-PRO蛋白、馬鈴薯X病毒編碼的Cmv2b蛋白、獸棚病毒編碼的B2蛋白等[64]。

RNA干擾也是抑制破壞基因結構的一種DNA片段轉錄子活性的重要方式。轉錄子通常以逆轉錄的方式在基因組中擴增。在逆轉錄過程中產生的雙鏈RNA分子可以被Dicer識別，從而被降解[63]。

目前發現[來源請求]，RNAi機制中的相關一些因子如內源性雙鏈RNA及蛋白因子可以在多種層次上對基因表達進行調控，其範圍已經超越了PTGS（post transcriptional gene silencing），如RNAi機制同樣參與了轉錄水平上的基因表達調控過程中。

Biological functions

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Immunity

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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

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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

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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

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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

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Gene knockdown

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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

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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

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參見：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

編輯

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

編輯

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

編輯

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

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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

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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

編輯

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

編輯

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
編輯

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
編輯

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

編輯

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]

歷史

編輯
 
以RNAi技術抑制矮牽牛花的色素基因。左邊的為野生種，右邊的為利用轉位子破壞色素基因的結果，中間的則為RNAi操作結果[151]

RNA干擾最早是在植物體身上發現[152]。1990年由約根森（Jorgensen）研究小組為得到顏色更深的矮牽牛花，而過量表達查爾酮合成酶（英語：chalcone synthase），以得到更多的粉紅色及紫色色素。結果意外得到了白色和白紫雜色的矮牽牛花，並且過量表達查爾酮合成酶的矮牽牛花中查爾酮合成酶的濃度比正常矮牽牛花中的濃度低50倍。約根森推測外源轉入的編碼查爾酮合成酶的基因同時抑制了花中內源查爾酮合成酶基因的表達[153]。後來研究者又在粉色麵包黴菌上發現了類似的現象[154]，但當時沒有人意會到兩者是同一現象。進一步研究發現，這些基因抑制現象都源自於mRNA的降解[155]，並稱此現象為「基因表現的共同抑制」（co-suppression of gene expression），但此時詳細機制並不清楚[156]。

不久之後，植物病毒學家在一個希望改良植物對於病毒抵抗力的實驗中，發現了類似的現象。當時已知植物會表現抗病毒蛋白，加強自身抵抗力。但並不知道這些病毒帶來的RNA短片段，居然也能夠使植物產生免疫力。因此，研究者推測將病毒基因插入植物基因體中應該也可以得到相同的免疫力[157]。同時，研究者也將植物基因送入病毒，使病毒感植物體。結果植物的基因被抑制，此現象稱為「病毒誘導基因沉默」（virus-induced gene silencing，VIGS）。而這一類類似的現象則統稱為「轉錄後基因沉默」（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，羅馬諾（Romano）和Macino也在粗糙鏈孢霉中發現了外源導入基因可以抑制具有同源序列的內源基因的表達[161]。1995年，Guo和Kemphues在線蟲中也發現了RNA干擾現象[162]。

1998年，安德魯·法厄（Andrew Z. Fire）等在秀麗隱杆線蟲（C.elegans）中進行反義RNA抑制實驗時發現，作為對照加入的雙鏈RNA相比正義或反義RNA顯示出了更強的抑制效果[1]。從與靶mRNA的分子量比考慮，加入的雙鏈RNA的抑制效果要強於理論上1:1配對時的抑制效果，因此推測在雙鏈RNA引導的抑制過程中存在某種擴增效應並且有某種酶活性參與其中。並且將這種現象命名為RNA干擾。

2006年，安德魯·法厄與克雷格·梅洛（Craig C. Mello）由於在RNAi機制研究中的貢獻獲得諾貝爾生理及醫學獎。

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書籍

編輯
  • 《新藥藥物靶標開發技術》，2006年版，高等教育出版社，ISBN 7-04-018953-4

期刊文章

編輯
  • 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.


外部連結

編輯
 
維基共享資源上的相關多媒體資源：Koala0090/sandbox6
 
維基學院中的相關研究或學習資源：Koala0090/sandbox6

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