RNA silencing

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RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). [1] RNA silencing mechanisms are conserved among most eukaryotes. [2] The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) [3] and its subspecies repeat associated small interfering RNA (rasiRNA). [4]

Contents

Background

RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression. [5] [6] [7] RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20–30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification. [8] [9] [10] In the context in which the phenomenon was first studied, small RNA was found to play an important role in defending plants against viruses. For example, these studies demonstrated that enzymes detect double-stranded RNA (dsRNA) not normally found in cells and digest it into small pieces that are not able to cause disease. [11] [12] [13] [14] [2]

While some functions of RNA silencing and its machinery are understood, many are not. For example, RNA silencing has been shown to be important in the regulation of development and in the control of transposition events. [15] RNA silencing has been shown to play a role in antiviral protection in plants as well as insects. [16] Also in yeast, RNA silencing has been shown to maintain heterochromatin structure. [17] However, the varied and nuanced role of RNA silencing in the regulation of gene expression remains an ongoing scientific inquiry. A range of diverse functions have been proposed for a growing number of characterized small RNA sequences—e.g., regulation of developmental, neuronal cell fate, cell death, proliferation, fat storage, haematopoietic cell fate, insulin secretion. [18]

RNA silencing functions by repressing translation or by cleaving messenger RNA (mRNA), depending on the amount of complementarity of base-pairing. RNA has been largely investigated within its role as an intermediary in the translation of genes into proteins. [19] More active regulatory functions, however, only began to be addressed by researchers beginning in the late-1990s. [20] The landmark study providing an understanding of the first identified mechanism was published in 1998 by Fire et al., [1] demonstrating that double-stranded RNA could act as a trigger for gene silencing. [20] Since then, various other classes of RNA silencing have been identified and characterized. [5] Presently, the therapeutic potential of these discoveries is being explored, for example, in the context of targeted gene therapy. [21] [22]

While RNA silencing is an evolving class of mechanisms, a common theme is the fundamental relationship between small RNAs and gene expression. [9] It has also been observed that the major RNA silencing pathways currently identified have mechanisms of action which may involve both post-transcriptional gene silencing (PTGS) [23] as well as chromatin-dependent gene silencing (CDGS) pathways. [5] CDGS involves the assembly of small RNA complexes on nascent transcripts and is regarded as encompassing mechanisms of action which implicate transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) events. [24] This is significant at least because the evidence suggests that small RNAs play a role in the modulation of chromatin structure and TGS. [25] [26]

Despite early focus in the literature on RNA interference (RNAi) as a core mechanism which occurs at the level of messenger RNA translation, others have since been identified in the broader family of conserved RNA silencing pathways acting at the DNA and chromatin level. [27] RNA silencing refers to the silencing activity of a range of small RNAs and is generally regarded as a broader category than RNAi. While the terms have sometimes been used interchangeably in the literature, RNAi is generally regarded as a branch of RNA silencing. To the extent it is useful to craft a distinction between these related concepts, RNA silencing may be thought of as referring to the broader scheme of small RNA related controls involved in gene expression and the protection of the genome against mobile repetitive DNA sequences, retroelements, and transposons to the extent that these can induce mutations. [28] The molecular mechanisms for RNA silencing were initially studied in plants [13] but have since broadened to cover a variety of subjects, from fungi to mammals, providing strong evidence that these pathways are highly conserved. [29]

At least three primary classes of small RNA have currently been identified, namely: small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA).

small interfering RNA (siRNA)

siRNAs act in the nucleus and the cytoplasm and are involved in RNAi as well as CDGS. [5] siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA (ssRNA) precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors. [30] The volume of literature on siRNA within the framework of RNAi is extensive. One of the potent applications of siRNAs is the ability to distinguish the target versus non-target sequence with a single-nucleotide difference. This approach has been considered as therapeutically crucial for the silencing dominant gain-of-function (GOF) disorders,where mutant allele causing disease is differed from wt-allele by a single nucleotide (nt). This type of siRNAs with capability to distinguish a single-nt difference are termed as allele-specific siRNAs. [31]

microRNA (miRNA)

The majority of miRNAs act in the cytoplasm and mediate mRNA degradation or translational arrest. [32] However, some plant miRNAs have been shown to act directly to promote DNA methylation. [33] miRNAs come from hairpin precursors generated by the RNaseIII enzymes Drosha and Dicer. [34] Both miRNA and siRNA form either the RNA-induced silencing complex (RISC) or the nuclear form of RISC known as RNA-induced transcriptional silencing complex (RITS). [35] The volume of literature on miRNA within the framework of RNAi is extensive.

Three prime untranslated regions and microRNAs

Three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

As of 2014, the miRBase web site, [36] an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). [37] Freidman et al. [37] estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. [38] Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold). [39] [40]

The effects of miRNA dysregulation of gene expression seem to be important in cancer. [41] For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes. [42]

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders. [43] [44] [45]

piwi-interacting RNA (piRNA)

piRNAs represent the largest class of small non-coding RNA molecules expressed in animal cells, deriving from a large variety of sources, including repetitive DNA and transposons. [46] However, the biogenesis of piRNAs is also the least well understood. [47] piRNAs appear to act both at the post-transcriptional and chromatin levels. They are distinct from miRNA due to at least an increase in terms of size and complexity. Repeat associated small interfering RNA (rasiRNAs) are considered to be a subspecies of piRNA. [4]

Mechanism

MiRNA processing MiRNA processing.svg
MiRNA processing

The most basic mechanistic flow for RNA Silencing is as follows: (For a more detailed explanation of the mechanism, refer to the RNAi:Cellular mechanism article.)

1: RNA with inverted repeats hairpin/panhandle constructs --> 2: dsRNA --> 3: miRNAs/siRNAs --> 4: RISC --> 5: Destruction of target mRNA

  1. It has been discovered that the best precursor to good RNA silencing is to have single stranded antisense RNA with inverted repeats which, in turn, build small hairpin RNA and panhandle constructs. [7] The hairpin or panhandle constructs exist so that the RNA can remain independent and not anneal with other RNA strands.
  2. These small hairpin RNAs and/or panhandles then get transported from the nucleus to the cytosol through the nuclear export receptor called exportin-5, and then get transformed into a dsRNA, a double stranded RNA, which, like DNA, is a double stranded series of nucleotides. If the mechanism didn't use dsRNAs, but only single strands, there would be a higher chance for it to hybridize to other "good" mRNAs. As a double strand, it can be kept on call for when it is needed.
  3. The dsRNA then gets cut up by a Dicer into small (21-28 nt = nucleotides long) strands of miRNAs (microRNAs) or siRNAs (short interfering RNAs.) A Dicer is an endoribonuclease RNase, which is a complex of a protein mixed with strand(s) of RNA.
  4. Lastly, the double stranded miRNAs/siRNAs separate into single strands; the antisense RNA strand of the two will combine with another endoribonuclease enzyme complex called RISC (RNA-induced silencing complex), which includes the catalytic component Argonaute, and will guide the RISC to break up the "perfectly complementary" target mRNA or viral genomic RNA so that it can be destroyed. [2] [7]
  5. It means that based on a short sequence specific area, a corresponding mRNA will be cut. To make sure, it will be cut in many other places as well. (If the mechanism only worked with a long stretch, then there would be higher chance that it would not have time to match to its complementary long mRNA.) It has also been shown that the repeated-associated short interference RNAs (rasiRNA) have a role in guiding chromatin modification. [2]

For an animated explanation of the mechanism of RNAi by Nature Reviews, see the External Links section below.

Biological functions

Immunity against viruses or transposons

RNA silencing is the mechanism that our cells (and cells from all kingdoms) use to fight RNA viruses and transposons (which originate from our own cells as well as from other vehicles). [2] In the case of RNA viruses, these get destroyed immediately by the mechanism cited above. In the case of transposons, it's a little more indirect. Since transposons are located in different parts of the genome, the different transcriptions from the different promoters produce complementary mRNAs that can hybridize with each other. When this happens, the RNAi machinery goes into action, debilitating the mRNAs of the proteins that would be required to move the transposons themselves. [48]

Down-regulation of genes

For a detailed explanation of the down-regulation of genes, see RNAi:downregulation of genes

Up-regulation of genes

For a detailed explanation of the up-regulation of genes, see RNAi:upregulation of genes

RNA silencing also gets regulated

The same way that RNA silencing regulates downstream target mRNAs, RNA silencing itself is regulated. For example, silencing signals get spread between cells by a group of enzymes called RdRPs (RNA-dependent RNA polymerases) or RDRs. [2]

Practical applications

Growing understanding of small RNA gene-silencing mechanisms involving dsRNA-mediated sequence-specific mRNA degradation has directly impacted the fields of functional genomics, biomedicine, and experimental biology. The following section describes various applications involving the effects of RNA silencing. These include uses in biotechnology, therapeutics, and laboratory research. Bioinformatics techniques are also being applied to identify and characterize large numbers of small RNAs and their targets.

Biotechnology

Artificial introduction of long dsRNAs or siRNAs has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms. [2] Structural and functional resolution of small RNAs as the effectors of RNA silencing has had a direct impact on experimental biology. For example, dsRNA may be synthesized to have a specific sequence complementary to a gene of interest. Once introduced into a cell or biological system, it is recognized as exogenous genetic material and activates the corresponding RNA silencing pathway. This mechanism can be used to effect decreases in gene expression with respect to the target, useful for investigating loss of function for genes relative to a phenotype. That is, studying the phenotypic and/or physiologic effects of expression decreases can reveal the role of a gene product. The observable effects can be nuanced, such that some methods can distinguish between “knockdown” (decrease expression) and “knockout” (eliminate expression) of a gene. [49] RNA interference technologies have been noted recently as one of the most widely utilized techniques in functional genomics. [50] Screens developed using small RNAs have been used to identify genes involved in fundamental processes such as cell division, apoptosis and fat regulation.

Biomedicine

Since at least the mid-2000s, there has been intensifying interest in developing short interfering RNAs for biomedical and therapeutic applications. [51] Bolstering this interest is a growing number of experiments which have successfully demonstrated the clinical potential and safety of small RNAs for combatting diseases ranging from viral infections to cancer as well as neurodegenerative disorders. [52] In 2004, the first Investigational New Drug applications for siRNA were filed in the United States with the Food and Drug Administration; it was intended as a therapy for age-related macular degeneration. [50] RNA silencing in vitro and in vivo has been accomplished by creating triggers (nucleic acids that induce RNAi) either via expression in viruses or synthesis of oligonucleotides. [53] Optimistically many studies indicate that small RNA-based therapies may offer novel and potent weapons against pathogens and diseases where small molecule/pharmacologic and vaccine/biologic treatments have failed or proved less effective in the past. [51] However, it is also warned that the design and delivery of small RNA effector molecules should be carefully considered in order to ensure safety and efficacy.

The role of RNA silencing in therapeutics, clinical medicine, and diagnostics is a fast developing area and it is expected that in the next few years some of the compounds using this technology will reach market approval. A report has been summarized below to highlight the many clinical domains in which RNA silencing is playing an increasingly important role, chief among them are ocular and retinal disorders, cancer, kidney disorders, LDL lowering, and antiviral. [53] The following table displays a listing of RNAi based therapy currently in various phases of clinical trials. The status of these trials can be monitored on the ClinicalTrials.gov website, a service of the National Institutes of Health (NIH). [54] Of note are treatments in development for ocular and retinal disorders, that were among the first compounds to reach clinical development. AGN211745 (sirna027) (Allergan) and bevasiranib (Cand5) (Opko) underwent clinical development for the treatment of age-related macular degeneration, but trials were terminated before the compounds reached the market. Other compounds in development for ocular conditions include SYL040012 (Sylentis) and QPI-007 (Quark). SYL040012 (bamosinan) is a drug candidate under clinical development for glaucoma, a progressive optic neurdegeneration frequently associated to increased intraocular pressure; QPI-007 is a candidate for the treatment of angle-closure glaucoma and Non-arteritic anterior ischaemic optic neuropathy; both compounds are currently undergoing phase II clinical trials. Several compounds are also under development for conditions such as cancer and rare diseases.

Clinical domainDrugIndicationTarget
Ocular and retinal disordersTD101Pachyonychia congenitaKeratin 6A N171K mutant
Ocular and retinal disordersQPI-1007Non-arteritic anterior ischaemic optic neuropathyCaspase 2
Ocular and retinal disordersAGN211745Age-related macular degeneration, choroidal neovascularizationVEGFR1
Ocular and retinal disordersPF-655Diabetic macular oedema, age-related macular degenerationRTP801
Ocular and retinal disordersSYL040012Glaucomaβ2 adrenergic receptor
Ocular and retinal disordersBevasiranibDiabetic macular oedemaVEGF
Ocular and retinal disordersBevasiranibMacular degenerationVEGF
CancerCEQ508Familial adenomatous polyposisβ-catenin
CancerALN-PLK1Liver tumorPLK1
CancerFANGSolid tumorFurin
CancerCALAA-01Solid tumorRRM2
CancerSPC2996chronic lymphocytic leukemiaBCL-2
CancerALN-VSP02Solid tumorVEGF, kinesin spindle protein
CancerNCT00672542Metastatic melanomaLMP2, LMP7, and MECL1
CancerAtu027Solid malignanciesPKN3
Kidney disordersQPI-1002/I5NPAcute kidney injuryp53
Kidney disordersQPI-1002/I5NPGraft dysfunction kidney transplantp53
Kidney disordersQPI-1002/I5NPKidney injury acute renal failurep53
LDL loweringTKM-ApoBHypercholesterolaemiaAPOB
LDL loweringPRO-040,201HypercholesterolaemiaAPOB
Antiviral miravirsen Hepatitis C virusmiR-122
AntiviralpHIV7-shI-TAR-CCR5RZHIVHIV Tat protein, HIV TAR RNA, human CCR5
AntiviralALN-RSV01RSVRSV nucleocapsid
AntiviralALN-RSV01RSV in lung transplant patientsRSV nucleocapsid

Main challenge

As with conventional manufactured drugs, the main challenge in developing successful offshoots of the RNAi-based drugs is the precise delivery of the RNAi triggers to where they are needed in the body. The reason that the ocular macular degeneration antidote was successful sooner than the antidote with other diseases is that the eyeball is almost a closed system, and the serum can be injected with a needle exactly where it needs to be. The future successful drugs will be the ones who are able to land where needed, probably with the help of nanobots. Below is a rendition of a table [53] that shows the existing means of delivery of the RNAi triggers.

Species/formulationPackaging capacityApplications and considerations
Viral vector
Adenovirus Usually < 10 KbdsDNA vector with large packaging capacity, transient expression, highly immunogenic
Adeno-associated virus (AAV)~4.5KbssDNA vector, small packaging capacity, mildly immunogenic, lasting expression in non-dividing cells, capsid pseudotyping/engineering facilitates specific cell-targeting
Lentivirus Up to 13.5 KbRNA vector, integration competent and incompetent forms available, less immunogenic than adenovirus or AAV, envelope pseudo typing facilitates cell targeting, clinical production more difficult than for adenovirus or AAV
Herpes simplexvirus 150 KbDNA vector, episomal, lasting expression, immunogenic
Bacterial vector species (bacterial minicells can carry plasmids, siRNAs or drugs)
Escherichis coli , S. TyphymuriumDelivery of short hairpin RNA or siRNA to gut tissue
Non-viral formulations
NanoparticleSelf-assembling, may target specific receptors, requires technical expertise to prepare
Stable nucleic acid lipid particle (SNALP)Stable for systemic delivery, broad cell-tye delivery
AptamerTargeting of specific receptors, requires sophisticated screening to develop
CholesterolStable for systemic delivery, broad cell-type delivery

Laboratory

The scientific community has been quick to harness RNA silencing as a research tool. The strategic targeting of mRNA can provide a large amount of information about gene function and its ability to be turned on and off. Induced RNA silencing can serve as a controlled method for suppressing gene expression. Since the machinery is conserved across most eukaryotes, these experiments scale well to a range of model organisms. [55] In practice, expressing synthetic short hairpin RNAs can be used to reach stable knock-down. [56] If promoters can be made to express these designer short hairpin RNAs, the result is often potent, stable, and controlled gene knock-down in both in vitro and in vivo contexts. [57] Short hairpin RNA vector systems can be seen as roughly analogous in scope to using cDNA overexpression systems. [58] Overall, synthetic and natural small RNAs have proven to be an important tool for studying gene function in cells as well as animals. [59]

Bioinformatics approaches to identify small RNAs and their targets have returned several hundred, if not thousands of, small RNA candidates predicted to affect gene expression in plants, C. elegans, D. melanogaster, zebrafish, mouse, rat, and human. [60] These methods are largely directed to identifying small RNA candidates for knock-out experiments but may have broader applications. One bioinformatics approach evaluated sequence conservation criteria by filtering seed complementary target-binding sites. The cited study predicted that approximately one third of mammalian genes were to be regulated by, in this case, miRNAs. [61]

Ethics & Risk-Benefit Analysis

One aspect of RNA silencing to consider is its possible off-target affects, toxicity, and delivery methods. If RNA silencing is to become a conventional drug, it must first pass the typical ethical issues of biomedicine. [62] Using risk-benefit analysis, researchers can determine whether RNA silencing conforms to ethical ideologies such as nonmaleficence, beneficence, and autonomy. [63]

There is a risk of creating infection-competent viruses that could infect non-consenting people. [64] There is also a risk of affecting future generations based on these treatments. These two scenarios, in respect to autonomy, is possible unethical. At this moment, unsafe delivery methods and unintended aspects of vector viruses add to the argument against RNA silencing. [63]

In terms of off-target effects, siRNA can induce innate interferon responses, inhibit endogenous miRNAs through saturation, and may have complementary sequences to other non-target mRNAs. These off-targets could also have target up-regulations such as oncogenes and antiapoptotic genes. The toxicity of RNA silencing is still under review as there are conflicting reports. [63] [64] [65]

Number of RNAi publications since 1998 RNAi publication statistics.png
Number of RNAi publications since 1998

RNA silencing is quickly developing, because of that, the ethical issues need to be discussed further. With the knowledge of general ethical principles, we must continuously perform risk-benefit analysis. [63]

See also

Related Research Articles

<span class="mw-page-title-main">Messenger RNA</span> RNA that is read by the ribosome to produce a protein

In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.

microRNA Small non-coding ribonucleic acid molecule

MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules by one or more of the following processes:

  1. Cleavage of the mRNA strand into two pieces,
  2. Destabilization of the mRNA by shortening its poly(A) tail, or
  3. Reducing translation of the mRNA into proteins.
<span class="mw-page-title-main">Gene expression</span> Conversion of a genes sequence into a mature gene product or products

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, and ultimately affect a phenotype. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. The process of gene expression is used by all known life—eukaryotes, prokaryotes, and utilized by viruses—to generate the macromolecular machinery for life.

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Regulation of gene expression</span> Modifying mechanisms used by cells to increase or decrease the production of specific gene products

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. It was discovered in 1998, by Andrew Fire at Carnegie Institution for Science in Washington DC and Craig Mello at University of Massachusetts in Worcester.

<span class="mw-page-title-main">Dicer</span> Enzyme that cleaves double-stranded RNA (dsRNA) into short dsRNA fragments

Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which functions in gene silencing via a variety of pathways at the transcriptional and translational levels. Using single-stranded RNA (ssRNA) fragments, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA), the complex functions as a key tool in gene regulation. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in defense against viral infections, as it is triggered by the presence of double-stranded RNA (dsRNA).

<span class="mw-page-title-main">Short hairpin RNA</span> Type of RNA

A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.

Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly.

<span class="mw-page-title-main">Argonaute</span> Protein that plays a role in RNA silencing process

The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the gene silencing phenomenon known as RNA interference (RNAi). Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity, which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay.

RNA activation (RNAa) is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the transcriptional/epigenetic level. RNAa was first reported in a 2006 PNAS paper by Li et al. who also coined the term "RNAa" as a contrast to RNA interference (RNAi) to describe such gene activation phenomenon. dsRNAs that trigger RNAa have been termed small activating RNA (saRNA). Since the initial discovery of RNAa in human cells, many other groups have made similar observations in different mammalian species including human, non-human primates, rat and mice, plant and C. elegans, suggesting that RNAa is an evolutionarily conserved mechanism of gene regulation.

<span class="mw-page-title-main">EIF2C1</span> Protein-coding gene in the species Homo sapiens

Protein argonaute-1 is a protein that in humans is encoded by the EIF2C1 gene.

Small activating Ribonucleic acids (saRNAs) are small double-stranded RNAs (dsRNAs) that target gene promoters to induce transcriptional gene activation in a process known as RNA activation (RNAa).

Gary Bruce Ruvkun is an American molecular biologist at Massachusetts General Hospital and professor of genetics at Harvard Medical School in Boston. Ruvkun discovered the mechanism by which lin-4, the first microRNA (miRNA) discovered by Victor Ambros, regulates the translation of target messenger RNAs via imperfect base-pairing to those targets, and discovered the second miRNA, let-7, and that it is conserved across animal phylogeny, including in humans. These miRNA discoveries revealed a new world of RNA regulation at an unprecedented small size scale, and the mechanism of that regulation. Ruvkun also discovered many features of insulin-like signaling in the regulation of aging and metabolism. He was elected a Member of the American Philosophical Society in 2019.

<span class="mw-page-title-main">RNA interference</span> Biological process of gene regulation

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.

DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell's endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs, that bring about the silencing of a target gene or genes once processed. Any RNA, including endogenous messenger RNA (mRNAs) or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target.

<span class="mw-page-title-main">CRISPR interference</span> Genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

<span class="mw-page-title-main">Microprocessor complex</span>

The microprocessor complex is a protein complex involved in the early stages of processing microRNA (miRNA) and RNA interference (RNAi) in animal cells. The complex is minimally composed of the ribonuclease enzyme Drosha and the dimeric RNA-binding protein DGCR8, and cleaves primary miRNA substrates to pre-miRNA in the cell nucleus. Microprocessor is also the smaller of the two multi-protein complexes that contain human Drosha.

References

  1. 1 2 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (Feb 1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature. 391 (6669): 806–11. Bibcode:1998Natur.391..806F. doi:10.1038/35888. PMID   9486653. S2CID   4355692.
  2. 1 2 3 4 5 6 7 Meister G, Tuschl T (Sep 2004). "Mechanisms of gene silencing by double-stranded RNA" (PDF). Nature. 431 (7006): 343–9. Bibcode:2004Natur.431..343M. doi:10.1038/nature02873. PMID   15372041. S2CID   90438.
  3. ↑ Monga I, Banerjee I (November 2019). "Computational Identification of piRNAs Using Features Based on RNA Sequence, Structure, Thermodynamic and Physicochemical Properties". Current Genomics. 20 (7): 508–518. doi:10.2174/1389202920666191129112705. PMC   7327968 . PMID   32655289.
  4. 1 2 Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, Nagami T, Siomi H, Siomi MC (Mar 2007). "A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila". Science. 315 (5818): 1587–90. Bibcode:2007Sci...315.1587G. doi: 10.1126/science.1140494 . PMID   17322028. S2CID   11513777.
  5. 1 2 3 4 Moazed D (Jan 2009). "Small RNAs in transcriptional gene silencing and genome defence". Nature. 457 (7228): 413–20. Bibcode:2009Natur.457..413M. doi:10.1038/nature07756. PMC   3246369 . PMID   19158787.
  6. ↑ Pickford AS, Cogoni C (May 2003). "RNA-mediated gene silencing". Cellular and Molecular Life Sciences. 60 (5): 871–82. doi:10.1007/s00018-003-2245-2. PMID   12827277. S2CID   5822771.
  7. 1 2 3 Tijsterman M, Ketting RF, Plasterk RH (2002). "The genetics of RNA silencing". Annual Review of Genetics. 36: 489–519. doi:10.1146/annurev.genet.36.043002.091619. PMID   12429701.
  8. ↑ Malecová B, Morris KV (Apr 2010). "Transcriptional gene silencing through epigenetic changes mediated by non-coding RNAs". Current Opinion in Molecular Therapeutics. 12 (2): 214–22. PMC   2861437 . PMID   20373265.
  9. 1 2 Meister G, Landthaler M, Dorsett Y, Tuschl T (Mar 2004). "Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing". RNA. 10 (3): 544–50. doi:10.1261/rna.5235104. PMC   1370948 . PMID   14970398.
  10. ↑ Zhou H, Hu H, Lai M (Dec 2010). "Non-coding RNAs and their epigenetic regulatory mechanisms". Biology of the Cell. 102 (12): 645–55. doi: 10.1042/BC20100029 . PMID   21077844. S2CID   11325463.
  11. ↑ Ding SW (Apr 2000). "RNA silencing". Current Opinion in Biotechnology. 11 (2): 152–6. doi:10.1016/s0958-1669(00)00074-4. PMID   10753772.
  12. ↑ Susi P, Hohkuri M, Wahlroos T, Kilby NJ (Jan 2004). "Characteristics of RNA silencing in plants: similarities and differences across kingdoms". Plant Molecular Biology. 54 (2): 157–74. doi:10.1023/B:PLAN.0000028797.63168.a7. PMID   15159620. S2CID   11018531.
  13. 1 2 Baulcombe D (Sep 2004). "RNA silencing in plants". Nature. 431 (7006): 356–63. Bibcode:2004Natur.431..356B. doi:10.1038/nature02874. PMID   15372043. S2CID   4421274.
  14. ↑ Baulcombe D (Jun 2005). "RNA silencing". Trends in Biochemical Sciences. 30 (6): 290–3. doi: 10.1016/j.tibs.2005.04.012 . PMID   15950871.
  15. ↑ Matzke MA, Birchler JA (Jan 2005). "RNAi-mediated pathways in the nucleus". Nature Reviews Genetics. 6 (1): 24–35. doi: 10.1038/nrg1500 . PMID   15630419. S2CID   9321759.
  16. ↑ Voinnet O (Mar 2005). "Induction and suppression of RNA silencing: insights from viral infections". Nature Reviews Genetics. 6 (3): 206–20. doi:10.1038/nrg1555. PMID   15703763. S2CID   26351712.
  17. ↑ Grewal SI, Rice JC (Jun 2004). "Regulation of heterochromatin by histone methylation and small RNAs". Current Opinion in Cell Biology. 16 (3): 230–8. doi:10.1016/j.ceb.2004.04.002. PMID   15145346.
  18. ↑ Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M (Nov 2004). "A pancreatic islet-specific microRNA regulates insulin secretion". Nature. 432 (7014): 226–30. Bibcode:2004Natur.432..226P. doi:10.1038/nature03076. PMID   15538371. S2CID   4415988.
  19. ↑ Eccleston, Alex; Angela K. Eggleston (2004). "RNA Interference". Nature. 431 (7006): 338–42. Bibcode:2004Natur.431..337E. doi: 10.1038/431337a . PMID   15372040.
  20. 1 2 Eggleston AK (Jan 2009). "RNA silencing". Nature. 457 (7228): 395. Bibcode:2009Natur.457..395E. doi: 10.1038/457395a . PMID   19158784.
  21. ↑ Takeshita F, Ochiya T (Aug 2006). "Therapeutic potential of RNA interference against cancer". Cancer Science. 97 (8): 689–96. doi: 10.1111/j.1349-7006.2006.00234.x . PMID   16863503. S2CID   19447542.
  22. ↑ Dykxhoorn DM, Novina CD, Sharp PA (Jun 2003). "Killing the messenger: short RNAs that silence gene expression". Nature Reviews Molecular Cell Biology. 4 (6): 457–67. doi:10.1038/nrm1129. PMID   12778125. S2CID   7445808.
  23. ↑ Hammond SM, Caudy AA, Hannon GJ (Feb 2001). "Post-transcriptional gene silencing by double-stranded RNA". Nature Reviews Genetics. 2 (2): 110–9. doi:10.1038/35052556. PMID   11253050. S2CID   2864720.
  24. ↑ Bühler M (Apr 2009). "RNA turnover and chromatin-dependent gene silencing". Chromosoma. 118 (2): 141–51. doi:10.1007/s00412-008-0195-z. PMID   19023586. S2CID   2790637.
  25. ↑ Gonzalez S, Pisano DG, Serrano M (Aug 2008). "Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs". Cell Cycle. 7 (16): 2601–8. doi: 10.4161/cc.7.16.6541 . PMID   18719372.
  26. ↑ Kim JK, Gabel HW, Kamath RS, Tewari M, Pasquinelli A, Rual JF, Kennedy S, Dybbs M, Bertin N, Kaplan JM, Vidal M, Ruvkun G (May 2005). "Functional genomic analysis of RNA interference in C. elegans". Science. 308 (5725): 1164–7. Bibcode:2005Sci...308.1164K. doi: 10.1126/science.1109267 . PMID   15790806. S2CID   15510615.
  27. ↑ Bühler M, Moazed D (Nov 2007). "Transcription and RNAi in heterochromatic gene silencing". Nature Structural & Molecular Biology. 14 (11): 1041–8. doi:10.1038/nsmb1315. PMID   17984966. S2CID   39098216.
  28. ↑ Dombroski BA, Feng Q, Mathias SL, Sassaman DM, Scott AF, Kazazian HH, Boeke JD (Jul 1994). "An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae". Molecular and Cellular Biology. 14 (7): 4485–92. doi:10.1128/mcb.14.7.4485. PMC   358820 . PMID   7516468.
  29. ↑ Svoboda P (2008). "RNA Silencing in Mammalian Oocytes and Early Embryos". RNA Interference. Current Topics in Microbiology and Immunology. Vol. 320. pp. 225–56. doi:10.1007/978-3-540-75157-1_11. ISBN   978-3-540-75156-4. PMID   18268847.
  30. ↑ Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, Xu J, Kittler EL, Zapp ML, Weng Z, Zamore PD (May 2008). "Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells". Science. 320 (5879): 1077–81. Bibcode:2008Sci...320.1077G. doi:10.1126/science.1157396. PMC   2953241 . PMID   18403677.
  31. ↑ Monga I, Qureshi A, Thakur N, Gupta AK, Kumar M (2017). "ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy". G3: Genes, Genomes, Genetics . 7 (9): 2931–2943. doi:10.1534/g3.117.044024. PMC   5592921 . PMID   28696921.
  32. ↑ Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS (Jun 2005). "Post-transcriptional gene silencing by siRNAs and miRNAs". Current Opinion in Structural Biology. 15 (3): 331–41. doi:10.1016/j.sbi.2005.05.006. PMID   15925505.
  33. ↑ Bao N, Lye KW, Barton MK (Nov 2004). "MicroRNA binding sites in Arabidopsis class III HD-ZIP mRNAs are required for methylation of the template chromosome". Developmental Cell. 7 (5): 653–62. doi: 10.1016/j.devcel.2004.10.003 . PMID   15525527.
  34. ↑ Zeng Y, Yi R, Cullen BR (Jan 2005). "Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha". The EMBO Journal. 24 (1): 138–48. doi:10.1038/sj.emboj.7600491. PMC   544904 . PMID   15565168.
  35. ↑ Irvine DV, Zaratiegui M, Tolia NH, Goto DB, Chitwood DH, Vaughn MW, Joshua-Tor L, Martienssen RA (Aug 2006). "Argonaute slicing is required for heterochromatic silencing and spreading". Science. 313 (5790): 1134–7. Bibcode:2006Sci...313.1134I. doi:10.1126/science.1128813. PMID   16931764. S2CID   42997104.
  36. ↑ miRBase.org
  37. 1 2 Friedman RC, Farh KK, Burge CB, Bartel DP (Jan 2009). "Most mammalian mRNAs are conserved targets of microRNAs". Genome Research. 19 (1): 92–105. doi:10.1101/gr.082701.108. PMC   2612969 . PMID   18955434.
  38. ↑ Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (Feb 2005). "Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs". Nature. 433 (7027): 769–73. Bibcode:2005Natur.433..769L. doi:10.1038/nature03315. PMID   15685193. S2CID   4430576.
  39. ↑ Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N (Sep 2008). "Widespread changes in protein synthesis induced by microRNAs". Nature. 455 (7209): 58–63. Bibcode:2008Natur.455...58S. doi:10.1038/nature07228. PMID   18668040. S2CID   4429008.
  40. ↑ Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP (Sep 2008). "The impact of microRNAs on protein output". Nature. 455 (7209): 64–71. Bibcode:2008Natur.455...64B. doi:10.1038/nature07242. PMC   2745094 . PMID   18668037.
  41. ↑ Palmero EI, de Campos SG, Campos M, de Souza NC, Guerreiro ID, Carvalho AL, Marques MM (Jul 2011). "Mechanisms and role of microRNA deregulation in cancer onset and progression". Genetics and Molecular Biology. 34 (3): 363–70. doi:10.1590/S1415-47572011000300001. PMC   3168173 . PMID   21931505.
  42. ↑ Bernstein C, Bernstein H (May 2015). "Epigenetic reduction of DNA repair in progression to gastrointestinal cancer". World Journal of Gastrointestinal Oncology. 7 (5): 30–46. doi: 10.4251/wjgo.v7.i5.30 . PMC   4434036 . PMID   25987950.
  43. ↑ Maffioletti E, Tardito D, Gennarelli M, Bocchio-Chiavetto L (2014). "Micro spies from the brain to the periphery: new clues from studies on microRNAs in neuropsychiatric disorders". Frontiers in Cellular Neuroscience. 8: 75. doi: 10.3389/fncel.2014.00075 . PMC   3949217 . PMID   24653674.
  44. ↑ Mellios N, Sur M (2012). "The Emerging Role of microRNAs in Schizophrenia and Autism Spectrum Disorders". Frontiers in Psychiatry. 3: 39. doi: 10.3389/fpsyt.2012.00039 . PMC   3336189 . PMID   22539927.
  45. ↑ Geaghan M, Cairns MJ (Aug 2015). "MicroRNA and Posttranscriptional Dysregulation in Psychiatry". Biological Psychiatry. 78 (4): 231–9. doi: 10.1016/j.biopsych.2014.12.009 . hdl: 1959.13/1335073 . PMID   25636176.
  46. ↑ Klattenhoff C, Theurkauf W (Jan 2008). "Biogenesis and germline functions of piRNAs". Development. 135 (1): 3–9. doi: 10.1242/dev.006486 . PMID   18032451.
  47. ↑ Ishizu H, Siomi H, Siomi MC (Nov 2012). "Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines". Genes & Development. 26 (21): 2361–73. doi:10.1101/gad.203786.112. PMC   3489994 . PMID   23124062.
  48. ↑ Matthew P Scott; Lodish, Harvey F.; Arnold Berk; Kaiser, Chris; Monty Krieger; Anthony Bretscher; Hidde Ploegh; Angelika Amon (2012). Molecular Cell Biology. San Francisco: W. H. Freeman. ISBN   978-1-4292-3413-9.
  49. ↑ Voorhoeve PM, Agami R (Jan 2003). "Knockdown stands up". Trends in Biotechnology. 21 (1): 2–4. doi:10.1016/S0167-7799(02)00002-1. PMID   12480342.
  50. 1 2 Karagiannis TC, El-Osta A (Oct 2005). "RNA interference and potential therapeutic applications of short interfering RNAs". Cancer Gene Therapy. 12 (10): 787–95. doi: 10.1038/sj.cgt.7700857 . PMID   15891770.
  51. 1 2 Kim DH, Rossi JJ (Mar 2007). "Strategies for silencing human disease using RNA interference". Nature Reviews Genetics. 8 (3): 173–84. doi:10.1038/nrg2006. PMID   17304245. S2CID   5781886.
  52. ↑ Stevenson M (Oct 2004). "Therapeutic potential of RNA interference". The New England Journal of Medicine. 351 (17): 1772–7. doi:10.1056/NEJMra045004. PMID   15496626. S2CID   1531568.
  53. 1 2 3 Davidson BL, McCray PB (May 2011). "Current prospects for RNA interference-based therapies". Nature Reviews Genetics. 12 (5): 329–40. doi:10.1038/nrg2968. PMC   7097665 . PMID   21499294.
  54. ↑ "ClinicalTrials.gov". clinicaltrials.gov.
  55. ↑ Zeng Y, Wagner EJ, Cullen BR (Jun 2002). "Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells". Molecular Cell. 9 (6): 1327–33. doi: 10.1016/s1097-2765(02)00541-5 . PMID   12086629.
  56. ↑ Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (Apr 2002). "Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells". Genes & Development. 16 (8): 948–58. doi:10.1101/gad.981002. PMC   152352 . PMID   11959843.
  57. ↑ Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW (Nov 2005). "Probing tumor phenotypes using stable and regulated synthetic microRNA precursors". Nature Genetics. 37 (11): 1289–95. doi:10.1038/ng1651. PMID   16200064. S2CID   15586239.
  58. ↑ Rigó G, Papdi C, Szabados L (2012). "Transformation Using Controlled cDNA Overexpression System". Plant Salt Tolerance. Methods in Molecular Biology. Vol. 913. pp. 277–90. doi:10.1007/978-1-61779-986-0_19. ISBN   978-1-61779-985-3. PMID   22895767.
  59. ↑ Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR, Sheth N, Bradshaw J, Burchard J, Kulkarni A, Cavet G, Sachidanandam R, McCombie WR, Cleary MA, Elledge SJ, Hannon GJ (Nov 2005). "Second-generation shRNA libraries covering the mouse and human genomes". Nature Genetics. 37 (11): 1281–8. doi:10.1038/ng1650. PMID   16200065. S2CID   17346898.
  60. ↑ Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E (Jan 2005). "Phylogenetic shadowing and computational identification of human microRNA genes". Cell. 120 (1): 21–4. doi: 10.1016/j.cell.2004.12.031 . PMID   15652478. S2CID   16403721.
  61. ↑ Lewis BP, Burge CB, Bartel DP (Jan 2005). "Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets". Cell. 120 (1): 15–20. doi: 10.1016/j.cell.2004.12.035 . PMID   15652477. S2CID   17316349.
  62. ↑ Beauchamp, TL; Childress, JF (2009). Principles of Biomedical Ethics, 6th ed. Oxford: Oxford University Press.
  63. 1 2 3 4 Ebbesen, Mette; Jensen, Thomas G.; Andersen, Svend; Pedersen, Finn Skou (2008-06-25). "Ethical Perspectives on RNA Interference Therapeutics". International Journal of Medical Sciences. 5 (3): 159–168. doi:10.7150/ijms.5.159. ISSN   1449-1907. PMC   2443345 . PMID   18612370.
  64. 1 2 Cullen, RC (2006). "Enhancing and Confirming the Specificity of RNAi Experiments". Nature Methods. 3 (9): 677–681. doi:10.1038/nmeth913. PMID   16929311. S2CID   13320443.
  65. ↑ Elbashir, SM; Martinez, J; Patkaniowska, A; Lendeckel, W; Tuschl, T (2001). "Functional Anatomy of siRNA for Mediating Efficient RNAi in Drosophilia melanogaster Embryo Lysate". EMBO Journal. 20 (23): 6877–88. doi:10.1093/emboj/20.23.6877. PMC   125328 . PMID   11726523.
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