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Defining the factors that contribute to on-target specificity of antisense oligonucleotides.

Lima WF, Vickers TA, Nichols J, Li C, Crooke ST - PLoS ONE (2014)

Bottom Line: The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent.Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system.These results provide a blueprint for design of more potent and more specific ASOs.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.

ABSTRACT
To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.

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Effect of E. coli RNase H overexpression on on-target antisense activity.HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).
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pone-0101752-g007: Effect of E. coli RNase H overexpression on on-target antisense activity.HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).

Mentions: To explore off-target activity in cells, the on-target sites for ASOs 38, 39, 82, and 83 were deleted from the SOD/TO minigene by site-directed mutagenesis. A stable 293 cell line harboring the mutated minigene construct, SOD 282_DL, and a second cell line that overexpresses E. coli RNase H (SOD 282_DLH) were treated with ASOs at concentrations between 0.5 and 150 nM. As a control, SOD/TO cells with and without RNase H overexpression were treated with the same ASOs (Fig. S5). All ASOs displayed similar levels of activity in the SOD/TO cell line with IC50s ranging from 5 to 15 nM (Fig. 7, solid lines). In the presence of E. coli RNase H, the potencies of the ASOs increased by 5–10 fold (Fig. 7, dashed lines). No activity was observed for any ASOs targeting the deleted region in the SOD 282_DL cells, suggesting that these sequences promote little or no off-target activity (Fig. 8, solid lines). However, in agreement with our nuclear extract results, off-target activity was observed for each of these ASOs when E. coli RNase H was overexpressed (Fig. 8, dashed lines). ASO 38 showed the greatest off-target activity under these conditions with an IC50 just 2-fold greater than that observed for the on-target activity in the presence of excess RNase H. The off-target activity of ASO 82 was approximately 3-fold less than that of 38 while the activity of ASOs 39 and 83 was 16 and 44-fold less respectively. Similar results were obtained when human RNase H1 was overexpressed in the same cell lines (Fig. S6), suggesting that the limiting levels of endogenous RNase H1 are a major contributor to ASO specificity in mammalian cells.


Defining the factors that contribute to on-target specificity of antisense oligonucleotides.

Lima WF, Vickers TA, Nichols J, Li C, Crooke ST - PLoS ONE (2014)

Effect of E. coli RNase H overexpression on on-target antisense activity.HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4114480&req=5

pone-0101752-g007: Effect of E. coli RNase H overexpression on on-target antisense activity.HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).
Mentions: To explore off-target activity in cells, the on-target sites for ASOs 38, 39, 82, and 83 were deleted from the SOD/TO minigene by site-directed mutagenesis. A stable 293 cell line harboring the mutated minigene construct, SOD 282_DL, and a second cell line that overexpresses E. coli RNase H (SOD 282_DLH) were treated with ASOs at concentrations between 0.5 and 150 nM. As a control, SOD/TO cells with and without RNase H overexpression were treated with the same ASOs (Fig. S5). All ASOs displayed similar levels of activity in the SOD/TO cell line with IC50s ranging from 5 to 15 nM (Fig. 7, solid lines). In the presence of E. coli RNase H, the potencies of the ASOs increased by 5–10 fold (Fig. 7, dashed lines). No activity was observed for any ASOs targeting the deleted region in the SOD 282_DL cells, suggesting that these sequences promote little or no off-target activity (Fig. 8, solid lines). However, in agreement with our nuclear extract results, off-target activity was observed for each of these ASOs when E. coli RNase H was overexpressed (Fig. 8, dashed lines). ASO 38 showed the greatest off-target activity under these conditions with an IC50 just 2-fold greater than that observed for the on-target activity in the presence of excess RNase H. The off-target activity of ASO 82 was approximately 3-fold less than that of 38 while the activity of ASOs 39 and 83 was 16 and 44-fold less respectively. Similar results were obtained when human RNase H1 was overexpressed in the same cell lines (Fig. S6), suggesting that the limiting levels of endogenous RNase H1 are a major contributor to ASO specificity in mammalian cells.

Bottom Line: The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent.Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system.These results provide a blueprint for design of more potent and more specific ASOs.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.

ABSTRACT
To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.

Show MeSH