scholarly journals 84. RNA Silencing: Role of Interferon System Components in Non_Specific Effects Induced by Chemically Synthesized Small Interfering RNAs, Enzymatically Synthesized Small Interfering RNAs and Short Hairpin RNAs

2006 ◽  
Vol 13 ◽  
pp. S35
Author(s):  
Christopher S. McAllister ◽  
Charles E. Samuel
Keyword(s):  
Rna Silencing ◽  
Small Interfering Rnas ◽  
System Components ◽  
Interferon System ◽  
Short Hairpin ◽  
Short Hairpin Rnas

scholarly journals Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Kazuya Terasawa ◽  
Kazuharu Shimizu ◽  
Gozoh Tsujimoto
Keyword(s):  
Biological Activity ◽  
Rna Interference ◽  
Gene Function ◽  
Small Interfering Rnas ◽  
Therapeutic Applications ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interferon Responses ◽  
Rnai Activity ◽  
Target Effects

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

scholarly journals Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats

2015 ◽  
Vol 59 (9) ◽  
pp. 5297-5305 ◽  
Author(s):  
Robert J. Scarborough ◽  
Kelsey L. Adams ◽  
Aïcha Daher ◽  
Anne Gatignol
Keyword(s):  
Target Site ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Gene Therapies ◽  
Sense Strand ◽  
Effective Inhibition ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Further Development ◽  
Hiv 1

ABSTRACTWe have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection.

scholarly journals Silencing of endogenous envelope genes in human choriocarcinoma cells shows that envPb1 is involved in heterotypic cell fusions

2012 ◽  
Vol 93 (8) ◽  
pp. 1696-1699 ◽  
Author(s):  
Lars Aagaard ◽  
Bolette Bjerregaard ◽  
Anders L. Kjeldbjerg ◽  
Finn Skou Pedersen ◽  
Lars-Inge Larsson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Health ◽  
Fusion Activity ◽  
Attractive Candidate ◽  
Short Hairpin ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Short Hairpin Rnas ◽  
Cell Fusions

Syncytin-1 and envPb1 are two conserved envelope genes in the human genome encoded by single loci from the HERV-W and -Pb families, respectively. To characterize the role of these envelope proteins in cell–cell fusion, we have developed lentiviral vectors that express short hairpin RNAs for stable knockdown of syncytin-1 and envPb1. Analysis of heterotypic fusion activity between trophoblast-derived choriocarcinoma BeWo cells, in which syncytin-1 and envPb1 are specifically silenced, and endothelial cells demonstrated that both syncytin-1 and envPb1 are important to fusion. The ability to fuse cells makes syncytin-1 and envPb1 attractive candidate molecules in therapy against cancer. Our available vectors may help eventually to decipher roles for these genes in human health and/or disease.

scholarly journals Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes

2017 ◽  
Author(s):  
James M. Burke ◽  
Rodney P. Kincaid ◽  
Francesca Aloisio ◽  
Nicole Welch ◽  
Christopher S. Sullivan
Keyword(s):  
Vector Space ◽  
Target Genes ◽  
Expression System ◽  
Rna Polymerase Iii ◽  
Mirna Gene ◽  
Small Interfering Rnas ◽  
Shrna Expression ◽  
Delivery Vector ◽  
Short Hairpin ◽  
Short Hairpin Rnas

AbstractShort hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, the U6 and H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by greater than two-fold as compared to the U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.

scholarly journals Trigger and suppression of antiviral defenses by grapevine Pinot gris virus (GPGV): novel insights into virus-host interaction

2021 ◽  
Author(s):  
Giulia Tarquini ◽  
Laura Pagliari ◽  
Paolo Ermacora ◽  
Rita Musetti ◽  
Giuseppe Firrao
Keyword(s):  
Rna Silencing ◽  
Infectious Clone ◽  
Transcriptional Gene Silencing ◽  
Antiviral Defense ◽  
Small Interfering Rnas ◽  
Host Interaction ◽  
Post Transcriptional Gene Silencing ◽  
Gene Expression Analyses ◽  
Grapevine Pinot Gris Virus

Grapevine Pinot gris virus (GPGV) is an emerging trichovirus that has been putatively associated with a novel grapevine disease known as grapevine leaf mottling and deformation (GLMD). Yet the role of GPGV in GLMD disease is poorly understood since it has been detected both in symptomatic and symptomless grapevines. We exploited a recently constructed GPGV infectious clone (pRI::GPGV-vir) to induce an antiviral response in Nicotiana benthamiana plants. In silico prediction of virus-derived small interfering RNAs (vsiRNAs) and gene expression analyses revealed the involvement of DCL4, AGO5 and RDR6 genes during GPGV infection, suggesting the activation of the post-transcriptional gene-silencing (PTGS) pathway as a plant antiviral defense. PTGS suppression assays in transgenic N. benthamiana 16c plants revealed the ability of the GPGV coat protein to suppress RNA silencing. This work provides novel insights on the interaction between GPGV and its host, revealing the ability of the virus to trigger and suppress antiviral RNA silencing.

Sequence, Chemical, and Structural Variation of Small Interfering RNAs and Short Hairpin RNAs and the Effect on Mammalian Gene Silencing

2003 ◽  
Vol 13 (2) ◽  
pp. 83-105 ◽  
Author(s):  
Jens Harborth ◽  
Sayda M. Elbashir ◽  
Kim Vandenburgh ◽  
Heiko Manninga ◽  
Stephen A. Scaringe ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Structural Variation ◽  
Small Interfering Rnas ◽  
Mammalian Gene ◽  
Short Hairpin ◽  
Short Hairpin Rnas

scholarly journals Artificial MicroRNA-Mediated Virus Resistance in Plants

2007 ◽  
Vol 81 (12) ◽  
pp. 6690-6699 ◽  
Author(s):  
Jing Qu ◽  
Jian Ye ◽  
Rongxiang Fang
Keyword(s):  
Virus Resistance ◽  
Rna Silencing ◽  
Plant Viruses ◽  
Short Hairpin Rna ◽  
Small Interfering Rnas ◽  
Hairpin Rna ◽  
Resistance Level ◽  
Artificial Mirna ◽  
Natural Defense ◽  
Short Hairpin

ABSTRACT RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.

scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

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