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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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Related in: MedlinePlus

Rescue of siRNA effectiveness by 5′ phosphorylation. Antisense oligonucleotide 2361 which has ANA modifications at its 5′end (and is therefore inactive) was synthesized including a 5′-terminal phosphate group and now termed 2516-P. NIH 3T3 MDR cells were treated with 50 nM siRNAs for 4 h, washed, and then analyzed for Pgp expression after 72 h. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells (means and standard errors of 3 determinations). (Inset) Assays after long exposures. The analysis was exactly the same as above except that the siRNAs and transfection agent were left in contact with the cells for the entire 76-h period.
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Figure 5: Rescue of siRNA effectiveness by 5′ phosphorylation. Antisense oligonucleotide 2361 which has ANA modifications at its 5′end (and is therefore inactive) was synthesized including a 5′-terminal phosphate group and now termed 2516-P. NIH 3T3 MDR cells were treated with 50 nM siRNAs for 4 h, washed, and then analyzed for Pgp expression after 72 h. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells (means and standard errors of 3 determinations). (Inset) Assays after long exposures. The analysis was exactly the same as above except that the siRNAs and transfection agent were left in contact with the cells for the entire 76-h period.

Mentions: Antisense strands with ANA modifications at the 5′ position were completely inactive, but the reason for this was not immediately apparent. There is a stabilizing effect of the ANA residue, thus potentially undermining the requirement for local instability at the 5′ antisense position (6); however, the same stabilizing effect would occur with modifications near the 3′ end of the sense strand, but these are well tolerated. Another possibility is that the 5′ ANA residue interferes with the required 5′ phosphorylation of the antisense strand of siRNA by endogenous kinases (5). To examine this possibility, we synthesized a version of an inactive oligonucleotide (2361) that has a 5′ phosphate group (termed 2516-P). As seen in Figure 5, the presence of a 5′-phosphate group partially rescues the effectiveness of the ANA-modified antisense strand. Oligonucleotides 2361 and 2516-P have exactly the same ANA modification at the 5′ position, but 2516-P was phosphorylated during synthesis. The observed ‘rescue’ of the ‘knockdown’ effect is not large but it is consistently observed. The degree of rescue was not affected by the presence or absence of serum during the transfection process (data not shown), suggesting that the relatively modest rescue is not because of the possible dephosphorylation of 2516-P by serum phosphatases, but rather that dephosphorylation and inactivation of 2516-P may take place within the cell. Interestingly, when we extended the period of transfection beyond the 4 h usually used, the effect of the 5′ phosphorylated ANA antisense was much greater, while the unphosphorylated ANA antisense remained inactive (Figure 5 inset). This suggests that maintaining a persistent external pool of 5′ phosphorylated siRNA, and thus continuous delivery, may allow adequate loading of the RISC complex even in the face of rapid dephosphorylation by cellular phosphatases.Figure 5.


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)

Rescue of siRNA effectiveness by 5′ phosphorylation. Antisense oligonucleotide 2361 which has ANA modifications at its 5′end (and is therefore inactive) was synthesized including a 5′-terminal phosphate group and now termed 2516-P. NIH 3T3 MDR cells were treated with 50 nM siRNAs for 4 h, washed, and then analyzed for Pgp expression after 72 h. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells (means and standard errors of 3 determinations). (Inset) Assays after long exposures. The analysis was exactly the same as above except that the siRNAs and transfection agent were left in contact with the cells for the entire 76-h period.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851637&req=5

Figure 5: Rescue of siRNA effectiveness by 5′ phosphorylation. Antisense oligonucleotide 2361 which has ANA modifications at its 5′end (and is therefore inactive) was synthesized including a 5′-terminal phosphate group and now termed 2516-P. NIH 3T3 MDR cells were treated with 50 nM siRNAs for 4 h, washed, and then analyzed for Pgp expression after 72 h. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells (means and standard errors of 3 determinations). (Inset) Assays after long exposures. The analysis was exactly the same as above except that the siRNAs and transfection agent were left in contact with the cells for the entire 76-h period.
Mentions: Antisense strands with ANA modifications at the 5′ position were completely inactive, but the reason for this was not immediately apparent. There is a stabilizing effect of the ANA residue, thus potentially undermining the requirement for local instability at the 5′ antisense position (6); however, the same stabilizing effect would occur with modifications near the 3′ end of the sense strand, but these are well tolerated. Another possibility is that the 5′ ANA residue interferes with the required 5′ phosphorylation of the antisense strand of siRNA by endogenous kinases (5). To examine this possibility, we synthesized a version of an inactive oligonucleotide (2361) that has a 5′ phosphate group (termed 2516-P). As seen in Figure 5, the presence of a 5′-phosphate group partially rescues the effectiveness of the ANA-modified antisense strand. Oligonucleotides 2361 and 2516-P have exactly the same ANA modification at the 5′ position, but 2516-P was phosphorylated during synthesis. The observed ‘rescue’ of the ‘knockdown’ effect is not large but it is consistently observed. The degree of rescue was not affected by the presence or absence of serum during the transfection process (data not shown), suggesting that the relatively modest rescue is not because of the possible dephosphorylation of 2516-P by serum phosphatases, but rather that dephosphorylation and inactivation of 2516-P may take place within the cell. Interestingly, when we extended the period of transfection beyond the 4 h usually used, the effect of the 5′ phosphorylated ANA antisense was much greater, while the unphosphorylated ANA antisense remained inactive (Figure 5 inset). This suggests that maintaining a persistent external pool of 5′ phosphorylated siRNA, and thus continuous delivery, may allow adequate loading of the RISC complex even in the face of rapid dephosphorylation by cellular phosphatases.Figure 5.

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
Related in: MedlinePlus