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Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters.

Watts JK, Yu D, Charisse K, Montaillier C, Potier P, Manoharan M, Corey DR - Nucleic Acids Res. (2010)

Bottom Line: Both guide and passenger strands can be modified and functional agRNAs can contain 2'F-RNA, 2'OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications.The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded.These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human progesterone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 2'F-RNA, 2'OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

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Native and modified duplexes have the same effect on antisense transcript levels. (A) Treatment of T47D cells with PR-9 and modified duplexes leads to a significant downregulation of both PR mRNA (left) and the promoter-associated antisense transcript (right). (B) Treatment of MCF7 cells with PR-11 and modified duplexes leads to a significant upregulation of PR mRNA (left) but no significant change in levels of the promoter-associated antisense transcript (right). *P < 0.05, **P < 0.01, ***P < 0.005.
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Figure 13: Native and modified duplexes have the same effect on antisense transcript levels. (A) Treatment of T47D cells with PR-9 and modified duplexes leads to a significant downregulation of both PR mRNA (left) and the promoter-associated antisense transcript (right). (B) Treatment of MCF7 cells with PR-11 and modified duplexes leads to a significant upregulation of PR mRNA (left) but no significant change in levels of the promoter-associated antisense transcript (right). *P < 0.05, **P < 0.01, ***P < 0.005.

Mentions: Native and modified duplexes gave comparable results. PR-9 and modified PR analogs reduced expression of both PR mRNA and the promoter-derived antisense transcript similarly (Figure 13A). Conversely, PR-11 and modified analogs increased expression of PR mRNA, but caused no significant change in antisense transcript levels (Figure 13B). The finding that native and modified duplexes have the same effect on levels of the antisense transcript supports the conclusion that they act through similar mechanisms.Figure 13.


Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters.

Watts JK, Yu D, Charisse K, Montaillier C, Potier P, Manoharan M, Corey DR - Nucleic Acids Res. (2010)

Native and modified duplexes have the same effect on antisense transcript levels. (A) Treatment of T47D cells with PR-9 and modified duplexes leads to a significant downregulation of both PR mRNA (left) and the promoter-associated antisense transcript (right). (B) Treatment of MCF7 cells with PR-11 and modified duplexes leads to a significant upregulation of PR mRNA (left) but no significant change in levels of the promoter-associated antisense transcript (right). *P < 0.05, **P < 0.01, ***P < 0.005.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 13: Native and modified duplexes have the same effect on antisense transcript levels. (A) Treatment of T47D cells with PR-9 and modified duplexes leads to a significant downregulation of both PR mRNA (left) and the promoter-associated antisense transcript (right). (B) Treatment of MCF7 cells with PR-11 and modified duplexes leads to a significant upregulation of PR mRNA (left) but no significant change in levels of the promoter-associated antisense transcript (right). *P < 0.05, **P < 0.01, ***P < 0.005.
Mentions: Native and modified duplexes gave comparable results. PR-9 and modified PR analogs reduced expression of both PR mRNA and the promoter-derived antisense transcript similarly (Figure 13A). Conversely, PR-11 and modified analogs increased expression of PR mRNA, but caused no significant change in antisense transcript levels (Figure 13B). The finding that native and modified duplexes have the same effect on levels of the antisense transcript supports the conclusion that they act through similar mechanisms.Figure 13.

Bottom Line: Both guide and passenger strands can be modified and functional agRNAs can contain 2'F-RNA, 2'OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications.The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded.These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human progesterone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 2'F-RNA, 2'OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

Show MeSH