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Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication.

ABSTRACT: Cell-free systems allow interference with gene expression processes without requiring elaborate genetic engineering procedures. This makes it ideally suited for rapid prototyping of synthetic biological parts. Inspired by nature's strategies for the control of gene expression via short antisense RNA molecules, we here investigated the use of small DNA (sDNA) for translational inhibition in the context of cell-free protein expression. We designed sDNA molecules to be complementary to the ribosome binding site (RBS) and the downstream coding sequence of targeted mRNA molecules. Depending on sDNA concentration and the promoter used for transcription of the mRNA, this resulted in a reduction of gene expression of targeted genes by up to 50-fold. We applied the cell-free sDNA technique (CF-sDNA) to modulate cell-free gene expression from the native T7 phage genome by suppressing the production of the major capsid protein of the phage. This resulted in a reduced phage titer, but at the same time drastically improved cell-free replication of the phage genome, which we utilized to amplify the T7 genome by more than 15 000-fold in a droplet-based serial dilution experiment. Our simple antisense sDNA approach extends the possibilities to exert translational control in cell-free expression systems, which should prove useful for cell-free prototyping of native phage genomes and also cell-free phage manipulation.

SUBMITTER: Vogele K

PROVIDER: S-EPMC7611488 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA) delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE) or human airway epithelia (HAE) grown at the air-liquid interface (ALI), the delivery of a Dicer-substrate small-interfering RNA (DsiRNA) duplex against hypoxanthine-guanine phosphoribosyltransferase (HPRT) with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2-5-day post-seeding) exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF), a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap) database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi) responses.Molecular Therapy - Nucleic Acids (2012) 1, e41; doi:10.1038/mtna.2012.36; published online 28 August 2012.

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Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication.

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