Milligram scale production of potent recombinant small interfering RNAs in Escherichia coli

Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double‐stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of “recombinant” siRNAs (pro‐siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro‐siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro‐siRNA production plasmid. Through the exploration of multiple pro‐siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro‐siRNA production plasmid. Together with a high–cell density fed‐batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro‐siRNA per liter of bacterial culture. The pro‐siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro‐siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under‐explored field.     

Reproducible features of small RNAs in C. elegans reveal NU RNAs and provide insights into 22G RNAs and 26G RNAs

Small RNAs regulate gene expression and most genes in the worm Caenorhabditis elegans are subject to their regulation. Here, we analyze small RNA data sets and use reproducible features of RNAs present in multiple data sets to discover a new class of small RNAs and to reveal insights into two known classes of small RNAs—22G RNAs and 26G RNAs. We found that reproducibly detected 22-nt RNAs, although are predominantly RNAs with a G at the 5′ end, also include RNAs with A, C, or U at the 5′ end. These RNAs are synthesized downstream from characteristic sequence motifs on mRNA and have U-tailed derivatives. Analysis of 26G RNAs revealed that they are processed from a blunt end of double-stranded RNAs and that production of one 26G RNA generates a hotspot immediately downstream for production of another. To our surprise, analysis of RNAs shorter than 18 nt revealed a new class of RNAs, which we call NU RNAs (pronounced “new RNAs”) because they have a NU bias at the 5′ end, where N is any nucleotide. NU RNAs are antisense to genes and originate downstream from U bases on mRNA. Although many genes have complementary NU RNAs, their genome-wide distribution is distinct from that of previously known classes of small RNAs. Our results suggest that current approaches underestimate reproducibly detected RNAs that are shorter than 18 nt, and theoretical considerations suggest that such shorter RNAs could be used for sequence-specific gene regulation in organisms like C. elegans that have small genomes.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

RNA干涉在提高植物营养价值上的应用

RNA干涉(RNAi)是由具同源性的外源dsRNA引起的序列特异性转录后基因沉默现象,广泛存在于各种动、植物中。人类大多数疾病,像心脏病和癌症,都能够通过食用添加具有特殊营养成分养的饮食来预防。应用RNAi技术可为人和动物提高植物的营养价值。     

Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA.     

Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics.     

Inhibition of the osteoclast V-ATPase by small interfering RNAs

The multisubunit enzyme V-ATPase harbours isoforms of individual subunits. a3 is one of four 116 kDa subunit a isoforms, and it is crucial for bone resorption. We used small interfering RNA (siRNA) molecules to knock down a3 in rat osteoclast cultures. Labeled siRNA-molecules entered osteoclasts via endocytosis and knocked down the a3 mRNA. Bone resorption was decreased in siRNA-treated samples due to decreased acidification and osteoclast inactivation. Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 in osteoclast cultures. Subunit a3 is thus an interesting target for novel nucleic acid therapy.     

Interference of hepatitis A virus replication by small interfering RNAs

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.     

Characterization of side reactions during the annealing of small interfering RNAs

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions—such as 2′,3′-isomerization, strand scission, and HF elimination—depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.     

Delivery of small interfering RNA with a synthetic collagen poly(Pro‐Hyp‐Gly) for gene silencing in vitro and in vivo

Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro‐Hyp‐Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long‐term gene silencing in vivo. We found that the SYCOL‐mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti‐luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL‐based non‐viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.     

Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

Reversal of BCRP-mediated multidrug resistance by stable expression of small interfering RNAs

Breast cancer resistance protein (BCRP) is an ATP-binding cassette multidrug transporter that confers resistance to various anticancer drugs like Mitoxantrone. Overexpression of BCRP confers multidrug resistance (MDR) in cancer cells and is a frequent impediment to successful chemotherapy. For stable reversal of BCRP-depending MDR by RNA interference technology, a hU6-RNA gene promoter-driven expression vector encoding anti-BCRP short hairpin RNA (shRNA) molecules was constructed. By treating endogenously and exogenously expresses high levels of BCRP cells with these constructs, expression of the targeted BCRP-encoding mRNA, and transport protein was inhibited completely. Furthermore, the accumulation of mitoxantrone in the anti-BCRP shRNA-treated cells increased. And the sensitivity to mitoxantrone of anti-BCRP shRNA-treated cells is increased 14.6-fold and 2.44-fold respectively compared to their control (P < 0.05). These data indicated that stable shRNA-mediated RNAi could be tremendously effective in reversing BCRP-mediated MDR and showed promises in overcoming MDR by gene therapeutic applications.     

Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome

India is endemic for foot-and-mouth disease and it continues to be a major threat to the livestock industry despite vaccination programmes. In the present study, the ability of specific small interfering (si)RNAs directed against different genomic regions of foot-and-mouth disease virus (FMDV) to inhibit virus replication in BHK-21 cells was examined. For preliminary evaluation of possible siRNA-mediated FMDV inhibition, a cocktail of several unique populations of 12-30bp siRNAs were successfully produced corresponding to three target regions located at structural (VP3-VP1), non-structural (2A-2C), and non-structural-untranslated (3D-3'UTR) region of serotype Asia1. Once the populations of siRNAs generated were found to reduce the virus titre significantly, two highly conserved 21bp siRNA duplexes were designed by analysing all FMDV sequence entries available in public-domain databases. In virus titration assay, more than 99% inhibition of virus yield for all the four serotypes (type Asia1, O, A, and C) could be demonstrated in cells transfected with each of the FMDV-specific siRNAs at 24h post-infection, compared to control cells transfected with scrambled siRNA. This was well supported by reduction in OD values in FMDV-specific sandwich ELISA. Although 100-fold reduction in virus titre with siRNA1 is substantial considering the transfection efficiency and fixed level of input siRNA, siRNA2 emerged to be a better choice as target where more than 300-fold reduction was observed and its inhibitory effect extended up to 48 h post-infection against all the serotypes. Interestingly, in the present study type A virus (IND 17/77) had a single mismatch at position 2 in the siRNA2 target region but it did not abrogate the inhibitory effect.     

Gene silencing in chick embryos with a vector-based small interfering RNA system

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.     

RNA干扰与siRNA（小干扰RNA）研究进展

RNA干扰作为后基因组时代的一种下调基因表达的工具已被广泛用于基因功能的研究以及疾病的治疗。在RNA干扰技术下调基因表达的背后存在着一个复杂的蛋白质网络参与了这一功能的实现。本文对近年来RNA干扰在疾病治疗方面的进展及其机制研究方面的进展作了一些概述。     

Development of a software tool and criteria evaluation for efficient design of small interfering RNA

RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds’s design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.