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Inhibition of MDR1 expression with altritol-modified siRNAs.

Fisher M, Abramov M, Van Aerschot A, Xu D, Juliano RL, Herdewijn P - Nucleic Acids Res. (2007)

Bottom Line: Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases.Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs.These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina Chapel Hill NC 27599, USA.

ABSTRACT
Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

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(A) Effects of ANA modifications in both strands of the duplex. NIH 3T3-MDR cells were treated with 50 nM siRNA and monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. A comparison is shown between siRNAs with ANA modifications in one strand (gray bars = modified in sense strand, white = modified in antisense strand) duplexed with a conventional RNA complementary strand, or siRNAs with ANA modifications in both strands (hatched bars). Results are the means of triplicate determinations. (B) Effects of mismatches. ANA-modified ORF2 siRNAs (50 nM) with (2531/2532) or without (2485/2487) 4 mismatches to the target sequence were tested for inhibition of Pgp expression versus control siRNA. The 2531/2532 (sense/antisense) mismatch duplex has ANAs in exactly the same positions as 2485/2487 (2532-uuc gUa uag GuC ucU aua* c*dtdt; mismatches indicated by capital letters, positions of ANAs indicated by *). Results are the means and standard errors of triplicate determinations.
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Figure 3: (A) Effects of ANA modifications in both strands of the duplex. NIH 3T3-MDR cells were treated with 50 nM siRNA and monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. A comparison is shown between siRNAs with ANA modifications in one strand (gray bars = modified in sense strand, white = modified in antisense strand) duplexed with a conventional RNA complementary strand, or siRNAs with ANA modifications in both strands (hatched bars). Results are the means of triplicate determinations. (B) Effects of mismatches. ANA-modified ORF2 siRNAs (50 nM) with (2531/2532) or without (2485/2487) 4 mismatches to the target sequence were tested for inhibition of Pgp expression versus control siRNA. The 2531/2532 (sense/antisense) mismatch duplex has ANAs in exactly the same positions as 2485/2487 (2532-uuc gUa uag GuC ucU aua* c*dtdt; mismatches indicated by capital letters, positions of ANAs indicated by *). Results are the means and standard errors of triplicate determinations.

Mentions: Use of two ANA-modified strands in a duplex usually provided additional advantages in terms of effectiveness (Figure 3A). For example, the modified sense strand 2359 and the modified antisense strand 2470 were both effective when duplexed with an unmodified partner; however, the 2359/2470 duplex was even more effective. Interestingly, duplexing a poorly effective antisense strand with an effective sense strand often partially ‘rescued’ siRNA activity; this can be seen in the 2359/2362 duplex in Figure 3A, suggesting that the overall characteristics of the duplex are important in RISC loading and mRNA degrading activity.Figure 3.


Inhibition of MDR1 expression with altritol-modified siRNAs.

Fisher M, Abramov M, Van Aerschot A, Xu D, Juliano RL, Herdewijn P - Nucleic Acids Res. (2007)

(A) Effects of ANA modifications in both strands of the duplex. NIH 3T3-MDR cells were treated with 50 nM siRNA and monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. A comparison is shown between siRNAs with ANA modifications in one strand (gray bars = modified in sense strand, white = modified in antisense strand) duplexed with a conventional RNA complementary strand, or siRNAs with ANA modifications in both strands (hatched bars). Results are the means of triplicate determinations. (B) Effects of mismatches. ANA-modified ORF2 siRNAs (50 nM) with (2531/2532) or without (2485/2487) 4 mismatches to the target sequence were tested for inhibition of Pgp expression versus control siRNA. The 2531/2532 (sense/antisense) mismatch duplex has ANAs in exactly the same positions as 2485/2487 (2532-uuc gUa uag GuC ucU aua* c*dtdt; mismatches indicated by capital letters, positions of ANAs indicated by *). Results are the means and standard errors of triplicate determinations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851637&req=5

Figure 3: (A) Effects of ANA modifications in both strands of the duplex. NIH 3T3-MDR cells were treated with 50 nM siRNA and monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. A comparison is shown between siRNAs with ANA modifications in one strand (gray bars = modified in sense strand, white = modified in antisense strand) duplexed with a conventional RNA complementary strand, or siRNAs with ANA modifications in both strands (hatched bars). Results are the means of triplicate determinations. (B) Effects of mismatches. ANA-modified ORF2 siRNAs (50 nM) with (2531/2532) or without (2485/2487) 4 mismatches to the target sequence were tested for inhibition of Pgp expression versus control siRNA. The 2531/2532 (sense/antisense) mismatch duplex has ANAs in exactly the same positions as 2485/2487 (2532-uuc gUa uag GuC ucU aua* c*dtdt; mismatches indicated by capital letters, positions of ANAs indicated by *). Results are the means and standard errors of triplicate determinations.
Mentions: Use of two ANA-modified strands in a duplex usually provided additional advantages in terms of effectiveness (Figure 3A). For example, the modified sense strand 2359 and the modified antisense strand 2470 were both effective when duplexed with an unmodified partner; however, the 2359/2470 duplex was even more effective. Interestingly, duplexing a poorly effective antisense strand with an effective sense strand often partially ‘rescued’ siRNA activity; this can be seen in the 2359/2362 duplex in Figure 3A, suggesting that the overall characteristics of the duplex are important in RISC loading and mRNA degrading activity.Figure 3.

Bottom Line: Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases.Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs.These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina Chapel Hill NC 27599, USA.

ABSTRACT
Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

Show MeSH