Production of highly potent recombinant siRNAs in Escherichia coli

Huang, Linfeng; Lieberman, Judy

doi:10.1038/nprot.2013.149

Cited by 18 publications

(12 citation statements)

References 34 publications

(34 reference statements)

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“…also demonstrated in vivo utility by inhibiting RNAi in a tissue-specific manner (54). Additionally, p19 has been used to stabilize siRNAs in bacteria for recombinant production of siRNA in E. coli (55). As these implementations all depend on tight binding to siRNA, the ultra-high affinity p19 clones reported here are expected to further enhance application performance and utility.…”

Section: Discussionmentioning

confidence: 99%

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

Yang

Kauke

Sun

et al. 2017

Nucleic Acids Research

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Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

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Section: Discussionmentioning

confidence: 99%

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

Yang

Kauke

Sun

et al. 2017

Nucleic Acids Research

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“…Many potent siRNAs already have been identified, and siRNAs have been shown to be producible in mammalian cells or E. coli , which makes large-scale production or RNAi expression libraries possible at low cost. 4 , 9 , 52 , 53 , 54 But these procedures are complicated and, due to the fact that in-vitro-produced siRNAs are not uniform, it will be difficult for them to be approved as drugs. 6 Here, we present a very simple procedure to produce small hairpin RNA as a special form of miR-451 mimics that can act as potent RNAi reagents as well as the innate immune response triggers.…”

Section: Discussionmentioning

confidence: 99%

A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

Sun

Riggs

2017

Molecular Therapy - Nucleic Acids

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We have studied the molecular properties of in-vitro-transcribed sliced small interfering RNAs (tsli-siRNAs) as an alternative RNAi agent for chemically synthesized siRNA. We describe here a simple and cost-effective procedure for high-purity production of tsli-siRNA using bacteriophage T7 RNA polymerases. tsli-siRNAs exhibit potent gene knockdown effects, with efficacy comparable with that of chemically synthesized sli-siRNAs and classical siRNAs. Furthermore, we found that it is very easy to prepare potent tsli-siRNAs with modified bases, such as 2′-fluorine- or biotin-16-modified tsli-siRNAs. tsli-siRNAs can cause a mild innate immune response, which can be easily eliminated by alkaline phosphatase treatment. On the other hand, this feature, which can be useful as a trigger of the innate immune response, can be enhanced by polynucleotide kinase treatment. Because of the simplicity of preparation and purification, the procedure presented here could be useful for the production of RNAi or immunostimulatory reagents.

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“…If siRNAs are to be used as lampricides, they will need to be produced in large quantities. They can already be produced chemically, but other simple and cost-effective methods of siRNA production have also been developed, including a readily-scalable, in vitro transcription method [56] and a microbial-expression system [57], where bacteria were used to produce siRNAs that were then mixed into food pellets and fed to shrimp [54].…”

Section: Discussionmentioning

confidence: 99%

RNA Interference Technology to Control Pest Sea Lampreys - A Proof-of-Concept

et al. 2014

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The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0–fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

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Production of highly potent recombinant siRNAs in Escherichia coli

Cited by 18 publications

References 34 publications

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

RNA Interference Technology to Control Pest Sea Lampreys - A Proof-of-Concept

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