Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells
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PROVIDER: PRJNA787253 | ENA |
REPOSITORIES: ENA
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Project description:Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for assessment of off-target effects of gapmer ASOs.
2021-12-08 | GSE190473 | GEO
Project description:NFYC-AS1 is an overlapping antisense RNA transcribed head-to-head to NFYC sense gene, encoding for the subunit C of NF-Y transcription factor, which is known as master regulator of cell cycle and proliferation in normal and tumor cells. Here we performed NFYC-AS1 silencing in lung squamous carcinoma H520 cells by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion. Afterwards, we performed differentially expressed analysis and gene set enrichement analysis to investigate on NFYC-AS1 function and mechanism of action.
2024-03-08 | GSE240468 | GEO
Project description:The experiment was carried out to provide insights into biological function of a newly identified long noncoding RNA, PNCTR. HeLa cells were transfected with either a PNCTR-specific GapmeR antisense oligonucleotide or a non-targeting GapmeR control. Total RNAs were extracted at 24 hours post transfection, QC'd and hybridized with oligo(dT) magnetic beads to isolate the poly(A) RNA fraction. Stranded mRNA sequencing libraries were then prepared using the Illumina TruSeq Stranded mRNA Library Kit and sequenced using an Illumina HiSeq 2500 instrument.
2018-10-12 | E-MTAB-6529 | biostudies-arrayexpress
Project description:RNA-seq analysis of HeLa cells treated with GapmeR antisense oligonucleotides
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Project description:Silencing of NFYC-AS1 by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion.
| PRJNA1003851 | ENA
Project description:Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off-target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off-target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off-target effects at the cellular level is not clear. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for the assessment of off-target effects of gapmer ASOs.
| S-EPMC6915909 | biostudies-literature
Project description:Using RNAseq we describe transcriptional changes downstream of MEK1 affecting RNA kncokdown following free uptake RNaseH1 gapmer antisense oligonucleotides
2021-04-22 | GSE172485 | GEO
Project description:Circular RNAs (circRNAs) constitute an abundant class of covalently closed non-coding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. Here, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We identified 5807 circRNAs in total RNA sequencing data from 4 liver cancer cell lines and used the back splice junction (BSJ) sequences of 7 validated circRNAs as target sites for designing different LNA-modified ASOs for circRNA knockdown. We found that while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript and may also cause degradation of unintended off-targets. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs or LNA/DNA mixmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes or predicted off-targets. In summary, our results demonstrate that LNA-modified ASOs complementary to BSJ sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.
2022-11-01 | GSE145592 | GEO
Project description:AimAntisense oligonucleotide (ASO) has the potential to induce off-target effects by inadvertent binding of ASOs to unintended RNAs that have a sequence similar to the target RNA. In the present study, we focused on the association between oligonucleotide length and off-target effects. Oligonucleotide extension is assumed to have bilateral effects on hybridization-dependent changes in gene expression, i.e., one is the decrease of off-target effects based on the reduced number of off-target candidate genes with perfect matches, and the other is the increase of off-target effects based on the increased binding affinity between the ASO and the complementary RNAs that leads to better tolerability for mismatches.MethodsTo determine the effects of oligonucleotide extension of gapmer ASOs on off-target effects, an extensive microarray analysis was performed using human cells treated with a 14-mer gapmer ASO and the extended 18-mer derivatives with the same core 14-mer region.Results and discussionOur data indicated that change in gene expression in the cells treated with 18-mer ASOs was significantly smaller than those with a 14-mer ASO, showing the decrease of off-target effects by oligonucleotide extension.
| S-EPMC8766371 | biostudies-literature
Project description:We investigated whether pharamacological inhibition of Smyd3 by antisense oligonucleotides (ASOs) can influence diethylnitrosamine (DEN)-induced liver cancer development. Our phenotypic analyses, among others, included gene expression profiling.
2020-06-24 | GSE153077 | GEO