Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1236218 | ENA |
REPOSITORIES: ENA
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Project description:Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.
2021-03-19 | GSE155764 | GEO
Project description:Whole blood rather than purified peripheral blood mononuclear cells is likely to become the prime tissue using expression microarrays for disease predication or prognosis however excess of globin mRNA may reduce probe detection sensitivity. In our study, we assessed whether whole-blood or globin-reduced RNA gives the most robust and sensitive results to detect small gene expression changes in response to hormone replacement therapy exposure. Each sample (N = 12) were hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using peptid nucleic acids (PNA) and using magnetic beads in the GlobinClear kit from Ambion. Finally, 7 and 4 technical replicates were conducted in no globin reduction and PNA groups, respectively. Both globin reduction approaches were mostly efficient at reducing globin RNA from cRNA. Samples processed by GlobinClear kit gave a very distinct gene expression profiles from the controls while samples processed with PNA gave an intermediary profile closest to the non globin reduction group with a slight increased sensitivity of transcript detection but a loss of reproducibility. Overall, no sign of higher sensitivity in detection of gene expression changes followed by hormone exposure was observed after globin reduction which was therefore judged not beneficial. Keywords: Groups comparaison To assess effect of globin reduction protocols on gene expression from whole-blood, we selected 12 postmenopausal women (6 HRT users and 6 non-HRT users) who were not using other medication than HRT at the time of blood sampling and in order to cover a wide body mass index (BMI) range in both HRT and non HRT users. The samples were also selected to contain the highest concentration of total extracted RNA. Each sample was hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using PNAs, globin reduction using magnetic beads in the GlobinClear™ kit from Ambion. The reproducibility of the globin reduction method compared to the non globin reduction approach was investigated for the PNA method only since this method was the most effective and specific. We planned to conduct 7 technical replicates in each group (i.e. no globin reduction and PNA groups). During amplification process with PNA, 4 samples failed to reverse transcript long fragment of mRNA certainly due to inhibitory contamination and these arrays were therefore excluded from our analyses. One sample (sample 34) was amplified with PNA twice the same day to study technical reproducibility without amplification date effect. Finally, a total of 47 arrays were conducted including 7 and 4 technical replicates in no globin reduction and PNA group, respectively.
2008-02-11 | E-GEOD-7008 | biostudies-arrayexpress
Project description:Here, we determined the ability of peptide nucleic acid (PNA) oligomers, coupled to different cell-penetrating peptides (CPPs), to interfere in regulatory RNA circuits of human blood-derived leukocytes. Using RNA-seq, FACS and confocal microscopy we identified octaarginin as a CPP enabling PNA delivery and sequence-dependent RNA inhibition in blood-derived myeloid cells at nanomolar concentration. At 200 nM, an R8-PNA targeting immune-regulatory microRNA-155 was delivered into nearly 100 % of human macrophages within 24 hours without apparent cytotoxicity, and globally de-repressed microRNA-155 target-mRNAs. This was not observed when coupling the PNA inhibitor to a K3 instead of the R8 peptide. We suggest that CPP choice is a fundamental success-determining factor for therapeutic RNA-inhibition in human myeloid leukocytes.
2021-03-01 | GSE152740 | GEO
Project description:Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.
2022-04-28 | GSE191313 | GEO