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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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ASO binding to the SOD-1 minigene mRNA spiked into denatured nuclear extract.(A) The binding profile of the ASOs for the mRNA in denatured extract (orange line) compared with the predicted target site accessibilities (green line). ASO binding is reported as percent untreated mRNA control (left y-axis). A greater mRNA reduction (lower percent control) correlates with tighter ASO binding. Target site accessibilities are reported as unpaired probabilities (right y-axis). Greater probabilities predict that the target region is single stranded and accessible to ASO, and lower probabilities suggest that the target region is involved in mRNA structure. (B) Prediction of ASO target site accessibility within the SOD-1 minigene mRNA using RNAP-fold. The scanning window size (w) and maximum allowed distance between the base-pairs (L) within the mRNA were set to, respectively, 80 and 40 ribonucleotides. The binding accessibility of each ASO for the target mRNA was calculated based on the length of the ASO target site (u), specifically 20 ribonucleotides.
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pone-0101752-g003: ASO binding to the SOD-1 minigene mRNA spiked into denatured nuclear extract.(A) The binding profile of the ASOs for the mRNA in denatured extract (orange line) compared with the predicted target site accessibilities (green line). ASO binding is reported as percent untreated mRNA control (left y-axis). A greater mRNA reduction (lower percent control) correlates with tighter ASO binding. Target site accessibilities are reported as unpaired probabilities (right y-axis). Greater probabilities predict that the target region is single stranded and accessible to ASO, and lower probabilities suggest that the target region is involved in mRNA structure. (B) Prediction of ASO target site accessibility within the SOD-1 minigene mRNA using RNAP-fold. The scanning window size (w) and maximum allowed distance between the base-pairs (L) within the mRNA were set to, respectively, 80 and 40 ribonucleotides. The binding accessibility of each ASO for the target mRNA was calculated based on the length of the ASO target site (u), specifically 20 ribonucleotides.

Mentions: ASO binding to the T7 transcribed SOD-1 minigene mRNA spiked into the denatured nuclear extract was determined using unlabeled SOD-1 minigene mRNA incubated in the denatured nuclear extract prior to the addition of the ASO and excess E. coli RNase H1 (Fig. S2B). Consistent with the naked 5′-32P labeled SOD-1 minigene mRNA, the E. coli RNase H1 cleavage activities for the mRNA added to the denatured nuclear extract varied significantly (Fig. 2 and 3A). Specifically, very little mRNA cleavage was observed for the 45 and 46 heteroduplexes. These experiments also showed that greater than 90% of the mRNA was full length and suggest that less than 10% of the mRNA was bound by these ASOs (Fig. 3A). Conversely, significantly greater cleavage activities were observed for ASOs 38 to 41 and 82 and 83. Incubation with these ASOs resulted in less than 10% full-length mRNA indicating that greater than 90% of the mRNA hybridized to ASO (Fig. 3A). One plausible explanation for the observed differences in the levels of heteroduplex formation is the higher order structure of the mRNA.


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)

ASO binding to the SOD-1 minigene mRNA spiked into denatured nuclear extract.(A) The binding profile of the ASOs for the mRNA in denatured extract (orange line) compared with the predicted target site accessibilities (green line). ASO binding is reported as percent untreated mRNA control (left y-axis). A greater mRNA reduction (lower percent control) correlates with tighter ASO binding. Target site accessibilities are reported as unpaired probabilities (right y-axis). Greater probabilities predict that the target region is single stranded and accessible to ASO, and lower probabilities suggest that the target region is involved in mRNA structure. (B) Prediction of ASO target site accessibility within the SOD-1 minigene mRNA using RNAP-fold. The scanning window size (w) and maximum allowed distance between the base-pairs (L) within the mRNA were set to, respectively, 80 and 40 ribonucleotides. The binding accessibility of each ASO for the target mRNA was calculated based on the length of the ASO target site (u), specifically 20 ribonucleotides.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4114480&req=5

pone-0101752-g003: ASO binding to the SOD-1 minigene mRNA spiked into denatured nuclear extract.(A) The binding profile of the ASOs for the mRNA in denatured extract (orange line) compared with the predicted target site accessibilities (green line). ASO binding is reported as percent untreated mRNA control (left y-axis). A greater mRNA reduction (lower percent control) correlates with tighter ASO binding. Target site accessibilities are reported as unpaired probabilities (right y-axis). Greater probabilities predict that the target region is single stranded and accessible to ASO, and lower probabilities suggest that the target region is involved in mRNA structure. (B) Prediction of ASO target site accessibility within the SOD-1 minigene mRNA using RNAP-fold. The scanning window size (w) and maximum allowed distance between the base-pairs (L) within the mRNA were set to, respectively, 80 and 40 ribonucleotides. The binding accessibility of each ASO for the target mRNA was calculated based on the length of the ASO target site (u), specifically 20 ribonucleotides.
Mentions: ASO binding to the T7 transcribed SOD-1 minigene mRNA spiked into the denatured nuclear extract was determined using unlabeled SOD-1 minigene mRNA incubated in the denatured nuclear extract prior to the addition of the ASO and excess E. coli RNase H1 (Fig. S2B). Consistent with the naked 5′-32P labeled SOD-1 minigene mRNA, the E. coli RNase H1 cleavage activities for the mRNA added to the denatured nuclear extract varied significantly (Fig. 2 and 3A). Specifically, very little mRNA cleavage was observed for the 45 and 46 heteroduplexes. These experiments also showed that greater than 90% of the mRNA was full length and suggest that less than 10% of the mRNA was bound by these ASOs (Fig. 3A). Conversely, significantly greater cleavage activities were observed for ASOs 38 to 41 and 82 and 83. Incubation with these ASOs resulted in less than 10% full-length mRNA indicating that greater than 90% of the mRNA hybridized to ASO (Fig. 3A). One plausible explanation for the observed differences in the levels of heteroduplex formation is the higher order structure of the mRNA.

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