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Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

Knockdown works in two ways. First, it improves the penetrating capability of water. It reduces the surface tension of plain water which allows it to penetrate surfaces where water might normally run off, to reach deep-seated fires.

This helps reduce the amount of water required to extinguish the fire and also provides quicker knockdown.

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In mammalian cells, short pieces of double-stranded RNA, otherwise known as short interfering RNA (siRNA), initiate the degradation or knockdown of a specific, targeted cellular mRNA. In this process, the antisense strand of the siRNA duplex becomes part of a multi-protein complex called the RNA-induced silencing complex (RISC). RISC then identifies the complementary mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, ultimately resulting in the loss of protein expression.

There are different methods for performing RNAi experiments. RNAi can be achieved by transfecting target cells with a pool of synthetic small RNAs or a pool of siRNA obtained via in vitro cleavage (in vitro dicing of target RNA). RNAi can also be achieved by transfecting cells with short hairpin RNA (shRNA) vectors. shRNA is processed within the cell, and the in vivo-generated siRNA can then target its specific mRNA molecule for degradation. Nontargeting controls can be used to test for specificity of the knockdown.

In the example below, Invitrogen Silencer Select siRNAs are transfected into cells in a 96-well format, followed by RT-qPCR. The most efficient knockdown (based on cell type and siRNA transfection conditions) is identified through gene expression analysis. The chosen screen is then scaled up for validation of antibody specificity through western blotting and immunocytochemistry (ICC).

Figure 7. Western blot for antibody validation.(A) Western blot showing knockdown of SMAD2 (lane 3) in HeLa whole cell lysates after transfection with SMAD2-targeting siRNA, using Invitrogen SMAD2 Recombinant Rabbit Monoclonal Antibody (31H15L4) (Cat. No. 700048). The knockdown is shown along with untreated and scrambled RNA as controls in lanes 1 and 2, respectively. Detection of actin was used as a loading control. (B) Relative quantitation of the knockdown of SMAD2 bands on the western blot when compared to the untreated and scrambled siRNA as the positive control. The intensity of each band is normalized using the relative intensities of the actin bands.

Figure 8. Immunocytochemistry for antibody validation. Knockdown of CHD7 was achieved by transfecting SH-SY5Y cells with CHD7-specific siRNA (Silencer select Cat. No. s31140, s529331). Immunofluorescence analysis was performed on untransfected SH-SY5Y cells (panel a, d), transfected with non-specific scrambled siRNA (panel b, e), and CHD7-specific siRNA (panel c, f). Cells were fixed, permeabilized, and labeled with CHD7 Polyclonal Antibody (Cat. No. PA5-72964, 1:100 dilution), followed by Goat anti-Rabbit IgG (Heavy Chain) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2,000). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962), and Rhodamine Phalloidin (Cat. No. R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of specific signal was observed upon siRNA-mediated knockdown (panels c, f) confirming specificity of the antibody to CHD7.

Advances in synthetic biology and metabolic engineering have enabled the efficient engineering of model bacteria for both biomedical1,2 and industrial3,4 applications. In the medical applications, a number of model probiotic bacteria (e.g., Escherichia coli Nissle 1917) have been engineered to carry out therapeutic1,5 or diagnostic6 tasks, but the vast majority of bacteria including human pathogens and commensal bacteria still lack tools for the systematic interrogation of gene expression and synthetic biology-based cell therapies. In the industrial biotechnology applications, the construction of efficient microbial cell factories to produce chemicals and materials from renewable resources relies on fine-tuning metabolic and regulatory networks4, but the identification and experimental validation of potential gene targets by chromosomal manipulation are labor-intensive and time-consuming. The use of trans-acting target gene knockdown systems such as CRISPR interference (CRISPRi) allows rapid knockdown of target genes at the transcriptional level without chromosomal manipulation7, and the recent Mobile-CRISPRi8 system can be used to knock down target genes in diverse bacteria. However, the practical applications of CRISPR-based tools in bacteria are sometimes limited due to the metabolic burden caused by the Cas9 protein9.

Here we report the development of a broad-host-range sRNA platform comprising sRNA scaffold and Hfq from Bacillus subtilis, with its versatility as a gene knockdown tool in diverse bacteria. As a medical application of this sRNA system, the virulence phenotypes are removed from pathogenic bacteria. Also, the sRNA system is applied to the metabolic engineering of different bacteria for the production of chemicals.

In these 14 different bacteria, the knockdown efficiency of BHR-sRNA system was tested using appropriate reporters. Due to the varying levels of difficulty in genetically manipulating these bacteria, three different strategies were employed. For S. epidermidis, R. opacus, C. xerosis, C. glutamicum BE, C. necator, V. natriegens, A. hydrophila, K. pneumoniae, and E. coli Nissle 1917, plasmids harboring genes encoding appropriate reporters (mRFPmars, EGFP, or GFP) were introduced to each strain by electroporation or conjugation (see Methods for details). For B. subtilis and P. putida, the EGFP gene was integrated into the respective chromosomes. For strains (L. lactis, S. coelicolor, and C. violaceum) where employing the two-plasmid system or chromosomal integration was difficult, knockdown of endogenous target genes that would result in phenotypic alterations was tested. In L. lactis, the upp gene (encoding uracil phosphoribosyltransferase) was selected as the knockdown target to examine the restoration of growth in the presence of toxic 5-fluorouracil31. S. coelicolor is known for its ability to produce the blue pigment actinorhodin, where knockdown of actIORFI encoding the ketosynthase of the minimal polyketide synthase would lead to the reduced production of actinorhodin32. C. violaceum produces bluish purple dyes violacein and deoxyviolacein, so knockdown of the first gene vioA in the violacein biosynthetic operon vioABCDE was tested33.

The BHR-sRNA system allowed successful knockdown of the reporter genes tested (Supplementary Table 5) in 15 out of 16 bacteria tested, with >50% of target gene repression achieved in six out of eight Gram-positive bacteria (S. epidermidis, B. subtilis, S. coelicolor, C. xerosis, C. glutamicum BE, and C. glutamicum ATCC 13032) and six out of eight Gram-negative bacteria (C. necator, P. putida, V. natriegens, A. hydrophila, K. pneumoniae, and E. coli DH5), demonstrating the broad applicability of the BHR-sRNA knockdown system in a wide range of bacteria (Fig. 2b, Supplementary Fig. 3a). For testing in L. lactis, only RoxS was used to knockdown the upp gene as the construction of the sRNA plasmid harboring BsHfq was unsuccessful.

We also tested the knockdown of the above reporter genes in 16 bacteria by employing the MicC-EcHfq system, and found that the BHR-sRNA system outperformed the MicC-EcHfq system in seven out of eight Gram-positive bacteria (L. lactis, S. epidermidis, B. subtilis, R. opacus, C. xerosis, C. glutamicum ATCC 13032, and C. glutamicum BE) and also in three out of eight Gram-negative bacteria (P. putida, K. pneumoniae, and E. coli DH5). The MicC-EcHfq system allowed >50% of target gene repression achieved in six bacteria (C. glutamicum ATCC 13032, S. coelicolor, E. coli DH5, C. necator, V. natriegens, and A. hydrophila). The generally improved knockdown effect of the BHR-sRNA system in Gram-positive bacteria might be explained by the evolutionary location of B. subtilis closer to many of the Gram-positive bacteria (Fig. 2a). In addition, the GC content of the RoxS scaffold (51.4%) is higher than that (43%) of the previously developed E. coli MicC scaffold which might have affected the scaffold stability, thus improving the knockdown effect in some Gram-negative bacteria (Supplementary Fig. 4). Further studies will be needed to understand the exact mechanisms affecting the knockdown efficiencies. ff782bc1db