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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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PR gene expression can be silenced by agRNAs containing combinations of modifications. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-9 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modification codes: dna, RNA, 2′F-RNA, 2′Ome-rna, lna; p = phosphate. (B) PR-9 analogs containing combinations of previously identified active strands. All data are normalized to mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. Protein levels were also confirmed by at least two independent transfections, from which a typical western blot is shown.
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Figure 8: PR gene expression can be silenced by agRNAs containing combinations of modifications. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-9 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modification codes: dna, RNA, 2′F-RNA, 2′Ome-rna, lna; p = phosphate. (B) PR-9 analogs containing combinations of previously identified active strands. All data are normalized to mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. Protein levels were also confirmed by at least two independent transfections, from which a typical western blot is shown.

Mentions: Many examples exist of potent and stable siRNAs containing multiple types of chemical modifications (38,54–56). In some cases, combination of multiple modifications led to potency and stability unattainable by the individual modifications alone (54,56). We set out to explore whether combination agRNA duplexes (Figure 8A) would yield better gene inhibition or activation in their respective cell lines. Even if existing potency is only maintained, the combination of different chemistries could afford better stability and other desirable drug properties.Figure 7.


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)

PR gene expression can be silenced by agRNAs containing combinations of modifications. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-9 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modification codes: dna, RNA, 2′F-RNA, 2′Ome-rna, lna; p = phosphate. (B) PR-9 analogs containing combinations of previously identified active strands. All data are normalized to mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. Protein levels were also confirmed by at least two independent transfections, from which a typical western blot is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926613&req=5

Figure 8: PR gene expression can be silenced by agRNAs containing combinations of modifications. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-9 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modification codes: dna, RNA, 2′F-RNA, 2′Ome-rna, lna; p = phosphate. (B) PR-9 analogs containing combinations of previously identified active strands. All data are normalized to mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. Protein levels were also confirmed by at least two independent transfections, from which a typical western blot is shown.
Mentions: Many examples exist of potent and stable siRNAs containing multiple types of chemical modifications (38,54–56). In some cases, combination of multiple modifications led to potency and stability unattainable by the individual modifications alone (54,56). We set out to explore whether combination agRNA duplexes (Figure 8A) would yield better gene inhibition or activation in their respective cell lines. Even if existing potency is only maintained, the combination of different chemistries could afford better stability and other desirable drug properties.Figure 7.

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