Abstract
Antisense oligonucleotides (ASOs) are most commonly designed to reduce targeted RNA via RNase H1-dependent degradation, however kinetic parameters for ASO-mediated targeting and subsequent cleavage and degradation of RNA in living cells are poorly understood. In this manuscript we use an inducible minigene system to determine the time course of ASO activity in the cell. Estimates of the time required for the ASO to enter and traverse the cell, scan the target mRNA, bind the cognate site, recruit RNase H1 and initiate cleavage, are presented in the context of transcription and mRNA processing rates. Data are also presented which indicate that rates for RNase H1-dependent ASO-mediated degradation of the targeted RNAs are different for nuclear-retained versus RNAs exported to the cytoplasm and that the level of RNase H1 in the cell and cellular compartments is limiting to the rate of ASO activity. In both cellular compartments RNase H1 ASOs essentially double the endogenous rates of clearance of the target RNA. Overexpression of Escherichia coli RNase H1 or the presence of multiple cognate sites each further increase the rate of target RNA degradation.
Highlights
Antisense oligonucleotide (ASO) mediated degradation of targeted RNAs has been broadly exploited as both a research tool and a platform to create human therapeutics [1]
All linkages were phosphorothioate with 2 -O-methoxyethyl (MOE) or constrained ethyl residues incorporated at the underlined positions
Cells were harvested at specific intervals following addition of TET, minigene expression assessed by qRT/polymerase chain reaction (PCR) using a primer/probe specific to the pre-mRNA
Summary
Antisense oligonucleotide (ASO) mediated degradation of targeted RNAs has been broadly exploited as both a research tool and a platform to create human therapeutics [1]. Human RNase H1 is active as a single peptide, whereas RNase H2 is a heterotrimeric enzyme [4,5]. Both enzymes are thought to play a role in DNA replication and repair, but additional biological functions are likely for both. Both RNase H isozymes recognize an RNA–
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