RNA interference
RNA interference (RNAi) is a biological process in which
Two types of small
The RNAi pathway is a naturally occurring process found in many
RNAi is a valuable research tool, both in
Cellular mechanism
RNAi is an RNA-dependent gene silencing process that is controlled by RISC and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component Argonaute.[6] When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by Dicer. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC.[7]
Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer,[8] which binds and cleaves dsRNAs in plants, or short hairpin RNAs (shRNAs) in humans, to produce double-stranded fragments of 20–25 base pairs with a 2-nucleotide overhang at the 3′ end.[9] Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects.[10] These short double-stranded fragments are called siRNAs. These siRNAs are then separated into single strands and integrated into an active RISC, by RISC-Loading Complex (RLC). RLC includes Dicer-2 and R2D2, and is crucial to unite Ago2 and RISC.[11] TATA-binding protein-associated factor 11 (TAF11) assembles the RLC by facilitating Dcr-2-R2D2 tetramerization, which increases the binding affinity to siRNA by 10-fold. Association with TAF11 would convert the R2-D2-Initiator (RDI) complex into the RLC.[12] R2D2 carries tandem double-stranded RNA-binding domains to recognize the thermodynamically stable terminus of siRNA duplexes, whereas Dicer-2 the other less stable extremity. Loading is asymmetric: the MID domain of Ago2 recognizes the thermodynamically stable end of the siRNA. Therefore, the "passenger" (sense) strand whose 5′ end is discarded by MID is ejected, while the saved "guide" (antisense) strand cooperates with AGO to form the RISC.[11]
After integration into the RISC,
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans and R2D2 in Drosophila, that stimulates Dicer activity.[15] The mechanism producing this length specificity is unknown and this protein only binds long dsRNAs.[15]
In C. elegans this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the Dicer-produced initiating or 'primary' siRNAs are used as templates.[16] These 'secondary' siRNAs are structurally distinct from Dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).[17][18]
MicroRNA
siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target.[23] In Drosophila and C. elegans, miRNA and siRNA are processed by distinct Argonaute proteins and Dicer enzymes.[24][25]
Three prime untranslated regions and microRNAs
Three prime untranslated regions (3′UTRs) of mRNAs often contain regulatory sequences that post-transcriptionally cause RNAi. Such 3′-UTRs often contain both binding sites for 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 2023, the miRBase web site,[26] an archive of miRNA sequences and annotations, listed 28,645 entries in 271 biologic species. Of these, 1,917 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).[27] Friedman et al.[27] 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.[28] 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).[29][30]
The effects of miRNA dysregulation of gene expression seem to be important in cancer.[31] For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.[32]
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.[33][34][35]
RISC activation and catalysis
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans and R2D2 in Drosophila, that stimulates Dicer activity.[15] This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown.[15] This RNA-binding protein then facilitates the transfer of cleaved siRNAs to the RISC complex.[36]
In C. elegans this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the Dicer-produced initiating or 'primary' siRNAs are used as templates.[16] These 'secondary' siRNAs are structurally distinct from Dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).[17][18]
The active components of an RNA-induced silencing complex (RISC) are
The structural basis for binding of RNA to the Argonaute protein was examined by
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
Transcriptional silencing
Components of the RNAi pathway are used in many eukaryotes in the maintenance of the organization and structure of their
The mechanism by which the RITS complex induces heterochromatin formation and organization is not well understood. Most studies have focused on the
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).[60] It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate.[61] Some pre-miRNAs do undergo A→I RNA editing[62][63] and this mechanism may regulate the processing and expression of mature miRNAs.[63] Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components.[64] 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.[65]
Variation among organisms
Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNAi 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
Some eukaryotic
Related prokaryotic systems
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.[74] It has been suggested that CRISPR interference systems in prokaryotes are analogous to eukaryotic RNAi systems, although none of the protein components are orthologous.[75]
Biological functions
This section needs to be updated.(May 2020) |
Immunity
RNAi is a vital part of the
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, RNAi is important in antiviral
The role of RNAi 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,[88][89] although this hypothesis has been challenged as poorly substantiated.[90] Evidence for the existence of a functional antiviral RNAi pathway in mammalian cells has been presented.[91][92]
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.[93]
Downregulation of genes
Endogenously expressed miRNAs, including both
Evolution
Based on
Applications
RNAi pathway for gene knockdown
Gene knockdown is a method used to reduce the expression of an organism’s specific genes. This is accomplished by using the naturally occurring process of RNAi.[6] This gene knockdown technique uses a double-stranded siRNA molecule that is synthesized with a sequence complementary to the gene of interest. The RNAi cascade begins once the Dicer enzyme starts to process siRNA. The end result of the process leads to degradation of mRNA and destroys any instructions needed to build certain proteins. Using this method, researchers are able to decrease (but not completely eliminate) the expression of a targeted gene. Studying the effects of this decrease in expression may show the physiological role or impact of the targeted gene products.[102][103]
Off-Target Effects of Gene Knockdown
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[104][105] mammal-specific,[106] and virus-specific[107] 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
Medications
The technique of knocking down genes using RNAi therapeutics has demonstrated success in randomized controlled clinical studies. These medications are a growing class of siRNA-based drugs that decrease the expression of proteins encoded by certain genes. To date, five RNAi medications have been approved by regulatory authorities in the US and Europe: patisiran (2018), givosiran (2019), lumasiran (2020), inclisiran (2020 in Europe with anticipated US approval in 2021), and vutrisiran (2022).[113][114][115][116]
While all of the current regulatory body approved RNAi therapeutics focus on diseases that originate in the liver, additional medications under investigation target a host of disease areas including cardiovascular diseases, bleeding disorders, alcohol use disorders, cystic fibrosis, gout, carcinoma, and eye disorders.
In 2019, the FDA and EMA approved
Lumasiran was approved as a siRNA-based medication in 2020 for use in both the European Union and the United States.[126][127] This medication is used for the treatment of primary hyperoxaluria type 1 (PH1) in pediatric and adult populations. The drug is designed to reduce hepatic oxalate production and urinary oxalate levels through RNAi by targeting hydroxyacid oxidase 1 (HAO1) mRNA for breakdown. Lowering HAO1 enzyme levels reduces the oxidation of glycolate to glyoxylate (which is a substrate for oxalate). Lumasiran is administered subcutaneously by a healthcare professional with dosing based on body weight.[128] Data from randomized controlled clinical trials indicate that the most common adverse reaction that was reported was injection site reactions. These reactions were mild and were present in 38 percent of patients treated with lumasiran.[129]
In 2022, the FDA and EMA approved vutrisiran for the treatment of adults with hereditary transthyretin mediated amyloidosis with polyneuropathy stage 1 or 2.[130][131] Vutrisiran is designed to break down the mRNA that codes for transthyretin.
Other investigational drugs using RNAi that are being developed by pharmaceutical companies such as Arrowhead Pharmaceuticals, Dicerna, Alnylam Pharmaceuticals, Amgen, and Sylentis. These medications cover a variety of targets via RNAi and diseases.
Investigational RNAi therapeutics in development:
Drug | Target | Delivery System | Disease | Phase | Status | Company | Identifier |
ALN–VSP02 | KSP and VEGF | LNP | Solid tumours | I | Completed | Alnylam Pharmaceuticals | NCT01158079 |
siRNA–EphA2–DOPC | EphA2 | LNP | Advanced cancers | I | Recruiting | MD Anderson Cancer Center | NCT01591356 |
Atu027 | PKN3 | LNP | Solid tumours | I | Completed | Silence Therapeutics | NCT00938574 |
TKM–080301 | PLK1 | LNP | Cancer | I | Recruiting | Tekmira Pharmaceutical | NCT01262235 |
TKM–100201 | VP24, VP35, Zaire Ebola L-polymerase | LNP | Ebola-virus infection | I | Recruiting | Tekmira Pharmaceutical | NCT01518881 |
ALN–RSV01 | RSV nucleocapsid | Naked siRNA | Respiratory syncytial virus infections | II | Completed | Alnylam Pharmaceuticals | NCT00658086 |
PRO-040201 | ApoB | LNP | Hypercholesterolaemia | I | Terminated | Tekmira Pharmaceutical | NCT00927459 |
ALN–PCS02 | PCSK9 | LNP | Hypercholesterolaemia | I | Completed | Alnylam Pharmaceuticals | NCT01437059 |
ALN–TTR02 | TTR | LNP | Transthyretin-mediated amyloidosis | II | Recruiting | Alnylam Pharmaceuticals | NCT01617967 |
CALAA-01 | RRM2 | Cyclodextrin NP | Solid tumours | I | Active | Calando Pharmaceuticals | NCT00689065 |
TD101 | K6a (N171K mutation) | Naked siRNA | Pachyonychia congenita | I | Completed | Pachyonychia Congenita Project | NCT00716014 |
AGN211745 | VEGFR1 | Naked siRNA | Age-related macular degeneration, choroidal neovascularization | II | Terminated | Allergan | NCT00395057 |
QPI-1007 | CASP2 | Naked siRNA | Optic atrophy, non-arteritic anterior ischaemic optic neuropathy | I | Completed | Quark Pharmaceuticals | NCT01064505 |
I5NP | p53 | Naked siRNA | Kidney injury, acute renal failure | I | Completed | Quark Pharmaceuticals | NCT00554359 |
Delayed graft function, complications of kidney transplant | I, II | Recruiting | Quark Pharmaceuticals | NCT00802347 | |||
PF-655 (PF-04523655) | RTP801 (Proprietary target) | Naked siRNA | Choroidal neovascularization, diabetic retinopathy, diabetic macular oedema | II | Active | Quark Pharmaceuticals | NCT01445899 |
siG12D LODER | KRAS | LODER polymer | Pancreatic cancer | II | Recruiting | Silenseed | NCT01676259 |
Bevasiranib | VEGF | Naked siRNA | Diabetic macular oedema, macular degeneration | II | Completed | Opko Health | NCT00306904 |
SYL1001 | TRPV1 | Naked siRNA | Ocular pain, dry-eye syndrome | I, II | Recruiting | Sylentis | NCT01776658 |
SYL040012 | ADRB2 | Naked siRNA | Ocular hypertension, open-angle glaucoma | II | Recruiting | Sylentis | NCT01739244 |
CEQ508 | CTNNB1 | Escherichia coli-carrying shRNA | Familial adenomatous polyposis | I, II | Recruiting | Marina Biotech | Unknown |
RXi-109 | CTGF | Self-delivering RNAi compound | Cicatrix scar prevention | I | Recruiting | RXi Pharmaceuticals | NCT01780077 |
ALN–TTRsc | TTR | siRNA–GalNAc conjugate | Transthyretin-mediated amyloidosis | I | Recruiting | Alnylam Pharmaceuticals | NCT01814839 |
ARC-520 | Conserved regions of HBV | DPC | HBV | I | Recruiting | Arrowhead Research | NCT01872065 |
Legal categorization and legal issues in a near future
Currently, both miRNA and SiRNA are currently chemically synthesized and so, are legally categorized inside EU and in USA as "simple" medicinal products. But as bioengineered siRNA (BERAs) are in development, these would be classified as biological medicinal products, at least in EU. The development of the BERAs technology raises the question of the categorization of drugs having the same mechanism of action but being produced chemically or biologically. This lack of consistency should be addressed.[132]
Delivery mechanisms
To achieve the clinical potential of RNAi, siRNA must be efficiently transported to the cells of target tissues. However, there are various barriers that must be fixed before it can be used clinically. For example, "naked" siRNA is susceptible to several obstacles that reduce its therapeutic efficacy.[133] Additionally, once siRNA has entered the bloodstream, naked RNA can be degraded by serum nucleases and can stimulate the innate immune system.[133] Due to its size and highly polyanionic (containing negative charges at several sites) nature, unmodified siRNA molecules cannot readily enter the cells through the cell membrane. Therefore, artificial or nanoparticle encapsulated siRNA must be used. If siRNA is transferred across the cell membrane, unintended toxicities can occur if therapeutic doses are not optimized, and siRNAs can exhibit off-target effects (e.g. unintended downregulation of genes with partial sequence complementarity).[134] Even after entering the cells, repeated dosing is required since their effects are diluted at each cell division. In response to these potential issues and barriers, two approaches help facilitate siRNA delivery to target cells: lipid nanoparticles and conjugates.[135]
Lipid nanoparticles
Lipid nanoparticles (LNPs) are based on liposome-like structures that are typically made of an aqueous center surrounded by a lipid shell.[136] A subset of liposomal structures used for delivery drugs to tissues rest in large unilamellar vesicles (LUVs) which may be 100 nm in size. LNP delivery mechanisms have become an increasing source of encasing nucleic acids and may include plasmids, CRISPR and mRNA.[137]
The first approved use of lipid nanoparticles as a drug delivery mechanism began in 2018 with the siRNA drug patisiran, developed by Alnylam Pharmaceuticals. Dicerna Pharmaceuticals, Persomics, Sanofi and Sirna Therapeutics also worked to bring RNAi therapies to market.[138][139]
Other recent applications include two FDA approved COVID-19 vaccines: mRNA-1273, developed by
Conjugates
In addition to LNPs, RNAi therapeutics have targeted delivery through siRNA conjugates (e.g., GalNAc, carbohydrates, peptides, aptamers, antibodies).[142] Therapeutics using siRNA conjugates have been developed for rare or genetic diseases such as acute hepatic porphyria (AHP), hemophilia, primary hyperoxaluria (PH) and hereditary ATTR amyloidosis as well as other cardiometabolic diseases such as hypertension and non-alcoholic steatohepatitis (NASH).[143]
Biotechnology
RNAi has been used for a variety of other applications including food, crops and insecticides. The use of the RNAi pathway has developed numerous products such as foods like Arctic apples, nicotine-free tobacco, decaffeinated coffee, nutrient fortified vegetation and hypoallergenic crops.[144][145][146] The emerging use of RNAi has the potential to develop many other products for future use.
Viral infection
Antiviral treatment is one of the earliest proposed RNAi-based medical applications, and two different types have been developed. The first type is to target viral RNAs. Many studies have shown that targeting viral RNAs can suppress the replication of numerous viruses, including
Cancer
While traditional chemotherapy can effectively kill cancer cells, lack of specificity for discriminating normal cells and cancer cells in these treatments usually cause severe side effects. Numerous studies have demonstrated that RNAi can provide a more specific approach to inhibit tumor growth by targeting cancer-related genes (i.e., oncogene).[158] It has also been proposed that RNAi can enhance the sensitivity of cancer cells to chemotherapeutic agents, providing a combinatorial therapeutic approach with chemotherapy.[159] Another potential RNAi-based treatment is to inhibit cell invasion and migration.[160]
Compared with chemotherapy or other anti-cancer drugs, there are a lot of advantages of siRNA drug.[161] SiRNA acts on the post-transcriptional stage of gene expression, so it does not modify or change DNA in a deleterious effect.[161] SiRNA can also be used to produce a specific response in a certain type of way, such as by downgrading suppression of gene expression.[161] In a single cancer cell, siRNA can cause dramatic suppression of gene expression with just several copies.[161] This happens by silencing cancer-promoting genes with RNAi, as well as targeting an mRNA sequence.[161]
RNAi drugs treat cancer by silencing certain cancer promoting genes.[161] This is done by complementing the cancer genes with the RNAi, such as keeping the mRNA sequences in accordance with the RNAi drug.[161] Ideally, RNAi is should be injected and/or chemically modified so the RNAi can reach cancer cells more efficiently.[161] RNAi uptake and regulation is controlled by the kidneys.[161]
Neurological diseases
RNAi strategies also show potential for treating
Stimulation of immune response
The human immune system is divided into two separate branches: the innate immune system and the adaptive immune system.[168] The innate immune system is the first defense against infection and responds to pathogens in a generic fashion.[168] On the other hand, the adaptive immune system, a system that was evolved later than the innate, is composed mainly of highly specialized B and T cells that are trained to react to specific portions of pathogenic molecules.[168]
The challenge between old pathogens and new has helped create a system of guarded cells and particles that are called safe framework.[168] This framework has given humans an army of systems that search out and destroy invader particles, such as pathogens, microscopic organisms, parasites, and infections.[168] The mammalian safe framework has developed to incorporate siRNA as a tool to indicate viral contamination, which has allowed siRNA is create an intense innate immune response.[168]
siRNA is controlled by the innate immune system, which can be divided into the acute inflammatory responses and antiviral responses.[168] The inflammatory response is created with signals from small signaling molecules, or cytokines.[168] These include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor α (TNF-α).[168] The innate immune system generates inflammation and antiviral responses, which cause the release pattern recognition receptors (PRRs).[168] These receptors help in labeling which pathogens are viruses, fungi, or bacteria.[168] Moreover, the importance of siRNA and the innate immune system is to include more PRRs to help recognize different RNA structures.[168] This makes it more likely for the siRNA to cause an immunostimulant response in the event of the pathogen.[168]
Food
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.
Development efforts have successfully reduced the levels of
Insecticides
RNAi is under development as an insecticide, employing multiple approaches, including genetic engineering and topical application.[4] Cells in the midgut of some insects take up the dsRNA molecules in the process referred to as environmental RNAi.[176] In some insects the effect is systemic as the signal spreads throughout the insect's body (referred to as systemic RNAi).[177]
Animals exposed to RNAi at doses millions of times higher than anticipated human exposure levels show no adverse effects.[178] RNAi has varying effects in different species of Lepidoptera (butterflies and moths).[179]
Transgenic plants
Topical
Alternatively dsRNA can be supplied without genetic engineering. One approach is to add them to
Functional genomics
Approaches to the design of genome-wide RNAi libraries can require more sophistication than the design of a single
Genome-scale screening
Genome-scale RNAi research relies on
History
RNAi discovery
The process of RNAi was referred to as "co-suppression" and "quelling" when observed prior to the knowledge of an RNA-related mechanism. The discovery of RNAi was preceded first by observations of transcriptional inhibition by
Not long after, plant
After these initial observations in plants, laboratories searched for this phenomenon in other organisms.
RNAi therapeutics
Just after Fire and Mello's ground-breaking discovery, Elbashir et al. discovered, by using synthetically made
The FDA approved the first siRNA-based drug (patisiran) in 2018. Givosiran and lumasiran later won FDA approval for the treatment of AHP and PH1 in 2019 and 2020, respectively.[113] Inclisiran received EMA approval in 2020 for the treatment of high cholesterol and is currently under review by the FDA.[208]
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External links
- Overview of the RNAi process, from Cambridge University's The Naked Scientists
- Animation of the RNAi process, from Nature
- NOVA scienceNOW explains RNAi – A 15-minute video of the PBS, 26 July 2005
- Silencing Genomes Archived 10 August 2019 at the Wayback Machine RNA interference (RNAi) experiments and bioinformatics in C. elegans for education. From the Dolan DNA Learning Center of Cold Spring Harbor Laboratory.
- RNAi screens in C. elegans in a 96-well liquid format and their application to the systematic identification of genetic interactions (a protocol)
- 2 American ‘Worm People’ Win Nobel for RNA Work, from NY Times
- Molecular Therapy web focus: "The development of RNAi as a therapeutic strategy", a collection of free articles about RNAi as a therapeutic strategy.
- GenomeRNAi: a database of phenotypes from RNA interference screening experiments in Drosophila melanogaster and Homo sapiens
- RNAi tools Archived 19 June 2018 at the Wayback Machine Pre-designed and custom RNA Interference tools