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 A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

2007 ◽  
Vol 8 (1) ◽  
pp. 98 ◽  
Author(s):  
Xiaocui Zhu ◽  
Leah A Santat ◽  
Mi Chang ◽  
Jamie Liu ◽  
Joelle R Zavzavadjian ◽  
...  
Keyword(s):  
Rna Interference ◽  
Multiple Gene ◽  
Short Hairpin ◽  
Short Hairpin Rnas

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 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.

RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter

2004 ◽  
Vol 385 (9) ◽  
pp. 791-794 ◽  
Author(s):  
Dorota Koper-Emde ◽  
Lutz Herrmann ◽  
Björn Sandrock ◽  
Bernd-Joachim Benecke
Keyword(s):  
Rna Interference ◽  
Gene Promoter ◽  
Target Sequence ◽  
Small Nuclear Rna ◽  
Small Interfering Rnas ◽  
Rna Duplexes ◽  
Inhibit Gene Expression ◽  
Short Hairpin Rnas ◽  
Rna Promoter

AbstractSmall interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more activein vivothan other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.

scholarly journals RNA interference and the use of small interfering RNA to study gene function in mammalian systems

2004 ◽  
Vol 33 (3) ◽  
pp. 545-557 ◽  
Author(s):  
I Bantounas ◽  
L A Phylactou ◽  
J B Uney
Keyword(s):  
Rna Interference ◽  
Gene Function ◽  
Viral Vector ◽  
Sirna Delivery ◽  
Interferon Response ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Rnai Technology

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

Target site effects in the RNA interference and microRNA pathways

2008 ◽  
Vol 36 (6) ◽  
pp. 1216-1219 ◽  
Author(s):  
Gregor Obernosterer ◽  
Hakim Tafer ◽  
Javier Martinez
Keyword(s):  
Rna Interference ◽  
Site Effects ◽  
Initial Study ◽  
Target Site ◽  
Small Interfering Rnas ◽  
Endonucleolytic Cleavage ◽  
Downstream Effects ◽  
Rna Targets ◽  
Target Sites ◽  
Rnai Activity

In RNAi (RNA interference), siRNAs (small interfering RNAs) are loaded into the RISC (RNA-induced silencing complex), which then mediates endonucleolytic cleavage of complementary target RNAs. Although RNAi has become one of the most powerful tools in molecular biology to assess gene function, there remains a great number of ineffective siRNAs. It is already known that the assembly and activation of RISC is a crucial determinant of RNAi activity, but downstream effects such as target accessibility have not been analysed extensively. Therefore we assessed the effect of target site accessibility and found that it significantly improves the potency of siRNAs. Similarly, miRNAs (microRNAs) act by repressing protein synthesis through imperfect base-pairing to the 3′-UTR (untranslated region) of target mRNAs. We found that predicted target sites reside in regions of high accessibility and tested whether this criterion could be used in the search of functional miRNA targets. In addition, we performed reporter gene assays to test whether accessibility correlates with measured mRNA suppression levels. The results of our initial study suggest that secondary structures might add a so far underrepresented layer of complexity in the recognition of RNA targets by miRNAs.

scholarly journals Minimal-length short hairpin RNAs: The relationship of structure and RNAi activity

RNA ◽  
2009 ◽  
Vol 16 (1) ◽  
pp. 106-117 ◽  
Author(s):  
Q. Ge ◽  
H. Ilves ◽  
A. Dallas ◽  
P. Kumar ◽  
J. Shorenstein ◽  
...  
Keyword(s):  
Minimal Length ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Relationship Of ◽  
The Relationship ◽  
Rnai Activity

scholarly journals Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

2020 ◽  
Author(s):  
Gonzalo Quiroga-Artigas ◽  
Alexandrea Duscher ◽  
Katelyn Lundquist ◽  
Justin Waletich ◽  
Christine E. Schnitzler
Keyword(s):  
Gene Function ◽  
Marine Invertebrates ◽  
Larval Settlement ◽  
Endogenous Gene ◽  
Successful Model ◽  
Hydractinia Symbiolongicarpus ◽  
Settlement And Metamorphosis ◽  
Short Hairpin ◽  
Visual Confirmation ◽  
Short Hairpin Rnas

AbstractPerforming gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

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