Artificial RNA Interference Research Articles

Precision RNAi using synthetic shRNAmir target sites.

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

Precision RNAi using synthetic shRNAmir target sites

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

Abstract 3973: Precision RNAi using synthetic shRNAmir target sites

Abstract Genetic loss-of-function methods including short-hairpin RNAs (shRNAs) and CRISPR are key methods for target gene validation across a variety of different disease areas. While these methods have revolutionized target gene discovery and characterization, both methods suffer from limitations in the context of in vivo target validation due to off-target effects and insufficient knock-down for shRNAs and resistant clones for CRISPR. In this study, we describe a method, artificial RNA interference (ARTi) that overcomes these limitations by fundamentally changing the basic experimental strategy of RNAi-based loss-of-function studies. Instead of newly designing gene-specific shRNA for individual target genes, ARTi utilizes ultra-effective and selective artificial miRNA-based shRNAs that do not match any transcribed gene in the target genome. In addition, these sequences are optimized for ultra-efficient miRNA processing and knockdown of synthetic target genes, and are deeply characterized to not trigger major off-target effects, enabling highly stringent target validation in vivo with unprecedented temporal control, selectivity and potency. We validate the approach by studying the in vitro and in vivo phenotypes of EGFR, KRAS and STAG1, genes relevant to cancer biology. This loss-of-function strategy will enable novel experimental strategies in therapeutic target validation and will be instrumental in guiding the lead optimization process by establishing genetic benchmark phenotypes. Citation Format: Ralph Neumüller, Thomas Hoffmann, Alexandra Hörmann, Maja Corcokovic, Johannes Zuber. Precision RNAi using synthetic shRNAmir target sites. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3973.

A modular RNA interference system for multiplexed gene regulation.

The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

Analysis of Variability in HIV-1 Subtype A Strains in Russia Suggests a Combination of Deep Sequencing and Multitarget RNA Interference for Silencing of the Virus.

Any method for silencing the activity of the HIV-1 retrovirus should tackle the extremely high variability of HIV-1 sequences and mutational escape. We studied sequence variability in the vicinity of selected RNA interference (RNAi) targets from isolates of HIV-1 subtype A in Russia, and we propose that using artificial RNAi is a potential alternative to traditional antiretroviral therapy. We prove that using multiple RNAi targets overcomes the variability in HIV-1 isolates. The optimal number of targets critically depends on the conservation of the target sequences. The total number of targets that are conserved with a probability of 0.7-0.8 should exceed at least 2. Combining deep sequencing and multitarget RNAi may provide an efficient approach to cure HIV/AIDS.

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE).

In our previous study, we generated a strain of 19-P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780-bp stem. We utilized this RNAi-induced strain to uncover RNAi-related genes. Random insertional mutagenesis was performed to generate tag-mutants that show a RNAi deficient phenotype. The 92-12C is one such tag-mutant, which bears a 14-kb deletion in chromosome 1. Complementation of 92-12C revealed that a protein gene, including a Cys-Cys-Cys-His-type zinc finger motif and an ankyrin repeat motif, is essential for effective RNAi in Chlamydomonas reinhardtii (Dangeard). BLAST analysis revealed that the zinc finger protein is homologous to an mRNA splicing-related protein of other species. Therefore, one of the probable scenarios is that mRNA coding for RNAi-related proteins cannot be properly spliced, which causes RNAi deficiency in the 92-12C tag-mutant.

Viruses, microRNAs and RNA Interference

In plants and invertebrates, RNA interference (RNAi) functions as a key innate immune response to viral infection. As a result, many plant viruses have evolved factors that can effectively inhibit RNAi and thereby facilitate virus replication. In contrast, efforts from several laboratories to identify an antiviral RNAi response in vertebrate cells infected by retro‐, flavi‐, or herpesviruses have so far failed to identify such a response, and artificial RNAi can be readily induced in virus infected human cells; i.e., viral infection of human cells does not result in a detectable inhibition of RNAi. In fact, rather than inhibiting RNAi, it is now clear that many DNA viruses encode microRNAs that are able to use the cellular RNAi machinery to inhibit the expression of cellular genes that have the potential to limit virus replication and/or that autoregulate viral gene expression. I will discuss our current understanding of the function of viral miRNAs and their potential contribution to viral pathogenesis, focusing on the identification of mRNA targets for the viral miRNAs encoded by the pathogenic human herpesviruses Herpes Simplex Virus 1 (HSV‐1) and Kaposi's Sarcoma‐associated Herpesvirus (KSHV).

Silencing of HIV-1 genes using siRNA-expressing genetic constructs

Today antiviral chemotherapy is the main methodto control HIV infection. Common medications influ-ence primarily the key enzymes of HIV-1, such asreverse transcriptase, integrase, and protease. How-ever, in spite of a large quantity of existing anti-HIVdrugs, antiviral therapy is often ineffective. The mainreason for ineffectiveness of treatment is adaptivemutagenesis of the virus, causing the appearance ofnew HIV-1 variants resistant to antiviral drugs. Thetoxicity and high cost of such drugs also present aserious problem.It is known that RNA interference is a powerfulnatural mechanism involved in the regulation of geneexpression and protection of cells against foreignRNA; it is also called ancient immunity operating atthe RNA level [1]. Recently, it has been found thatRNA interference is regulated during the developmentof an organism [2]. Viruses have the capacity to regu-late RNA interference. In the course of evolution, theyacquired the ability to turn off RNA interference in thehost cell to ensure their own reproduction [3]. Severalyears ago, we proposed artificial RNA interference forboth the investigation of its mechanisms and the use ofsilencing of HIV-1 genes in gene therapy. Now severalresearch groups are working in this field [4–6]. In thispaper, we report the results of testing three geneticconstructs for suppression of HIV-1 production inhuman cells.We induced artificial RNA interference using shorthairpin genetic constructs. Such constructs containshort palindromic sequences separated by a shortloop. The palindromic regions of RNA moleculesform hairpins. Owing to the existing cell mechanismof RNA monitoring, such double-stranded RNAs(dsRNAs) are detected and processed by Dicer(RNase III) to yield double-stranded small interferingRNA (siRNA). At the second stage of RNA interfer-ence, only the antisense siRNA strand remains withinthe RNA-induced silencing complex (RISC). A nucle-otide sequence of the siRNA within RISC is used tosearch for complementary regions in cell RNAs. In thecase of an HIV-1-specific siRNA, RISC recognizes anavailable single-stranded region in viral transcriptsand cuts it.The efficiency of siRNA is determined by a num-ber of factors. In particular, it depends on the siRNAnucleotide composition, which should meet the fol-lowing empiric requirements [7, 8]: a GC content of30–52%, at least three A/U in positions 15–19 of thesense strand, lack of internal repeats, A in positions 19and 3 of the sense strand, U in position 10 of the sensestrand, lack of G/C in position 19 of the sense strand,lack of G in position 13 of the sense strand, etc. Theavailability of target RNA is the second factor deter-mining the efficiency of siRNA. If the mRNA regioncomplementary to siRNA is unavailable (for example,it is bound to a protein or included in a duplex), thenit cannot be degraded.We began our study when these requirements wereunknown, so we proceeded from the assumptions thatthe best targets in HIV-1 transcripts are the most con-served regions within the functionally importantdomains and that biologically active siRNAs can beisolated in direct tests for suppression of virus produc-tion.We searched for HIV-1 conserved regions with theuse of the BLAST program and multiple sequencealignment. Within the AF316544 sequence of HIV-1subtype A, we found the most conserved regions cor-responding to the protease, reverse transcriptase, andTat domains. These regions were chosen as targets forsiRNA-encoding constructs. The resulting siRNAs

RNA interference for mammalian cells

Knock-out of mammalian genes is technically troublesome and time-consuming compared to those of lower animals. RNA interference (RNAi) is a strategy of sequence-specific post-translational gene silencing, and it has been successfully applied for disruption of gene transcripts in C. elegans. Gene silencing by RNAi had not been accomplished in mammalian cells until recently, because bystander activation of DNA-dependent protein kinase (PKR) coincides to disturb gene silencing by long double-stranded RNA (dsRNA), resulting in non-specific repression of translation of many proteins. A breakthrough has come recently with a report suggesting that 20- or 21-bp duplex RNA with 2 bp 3' overhang are made by DICER protein that cleaves long dsRNA during RNAi reactions in vivo and the resultant short dsRNAs mediate RNAi. Those short dsRNAs, namely, small interference RNA (siRNA), barely activate PKR. Using synthesized 21 bp siRNA, T. Tuschl's group has challenged to establish an artificial RNAi method suitable for mammalian cells. Their report was favorable in that siRNA specifically suppressed targeted gene translation in mammalian cells during culture without activation of PKR. Recently K. Taira's group developed the vector-based siRNA expression system by which RNAi is feasible in mammalian cells. Almost all genes can be targeted by RNAi. RNAi methods require minimal time and labor; therefore, mammalian gene knockdown by RNAi will become popular in the near future.