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Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.

Dutkiewicz M, Ojdowska A, Kuczynski J, Lindig V, Zeichhardt H, Kurreck J, Ciesiołka J - PLoS ONE (2015)

Bottom Line: Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro.For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells.Addition of the helper oligomer improved silencing capacity of the respective siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.

No MeSH data available.


Related in: MedlinePlus

Induction of RNase H cleavage by potential helper oligomers.(A) Nucleotide sequence of the 5’UTRcvb3 RNA. Sites mapped as accessible to oligonucleotide hybridization are indicated with gray rectangles. Target sites of DNA 16-mers, potential helper oligomers, are marked with black lines below the sequence and numbered 1–19. (B) Representative agarose gels showing degradation of 5’UTRcvb3 RNA by RNase H in the presence of one of the DNA 16-mers. Numbers correspond to target sites indicated in the sequence in (A). Ctr—control reaction without DNA oligonucleotide.
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pone.0136395.g003: Induction of RNase H cleavage by potential helper oligomers.(A) Nucleotide sequence of the 5’UTRcvb3 RNA. Sites mapped as accessible to oligonucleotide hybridization are indicated with gray rectangles. Target sites of DNA 16-mers, potential helper oligomers, are marked with black lines below the sequence and numbered 1–19. (B) Representative agarose gels showing degradation of 5’UTRcvb3 RNA by RNase H in the presence of one of the DNA 16-mers. Numbers correspond to target sites indicated in the sequence in (A). Ctr—control reaction without DNA oligonucleotide.

Mentions: To design helper antisense oligomers which would be able to destabilize the double-stranded stems of the targeted RNA, we tested the binding efficiency of several DNA 16-mers complementary to the 5’UTRcvb3 using the RNase H assay. Nineteen DNA 16-mers were chosen for the test based on our previous mapping results and in several cases the oligomers were shifted in the 3’ direction by 2, 3 or 4 nucleotides (Fig 3A). It turned out that several oligonucleotides were capable of inducing efficient RNase H cleavage upon binding: these were oligomers Nos. 1, 2 and 4–9. The upper band on the gel corresponding to the full-length RNA was degraded in those cases (Fig 3B). The 16-mers most efficient at inducing RNase H activity, were oligomers Nos. 2, 4 and 5, complementary to domain II. Unexpectedly, three other oligomers Nos. 11, 14 and 18 were completely inactive in inducing RNA degradation, although they were directed into sites mapped previously as accessible to hybridization. Eight other oligomers Nos. 3, 10, 12, 13, 15–17, and 19, were also inefficient in inducing RNase H activity. These results highlight the need for testing oligomers of the final length for the ability to bind RNA regions mapped as accessible with short-oligomer-libraries. The sequences of all oligomers are shown in S2 Table.


Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.

Dutkiewicz M, Ojdowska A, Kuczynski J, Lindig V, Zeichhardt H, Kurreck J, Ciesiołka J - PLoS ONE (2015)

Induction of RNase H cleavage by potential helper oligomers.(A) Nucleotide sequence of the 5’UTRcvb3 RNA. Sites mapped as accessible to oligonucleotide hybridization are indicated with gray rectangles. Target sites of DNA 16-mers, potential helper oligomers, are marked with black lines below the sequence and numbered 1–19. (B) Representative agarose gels showing degradation of 5’UTRcvb3 RNA by RNase H in the presence of one of the DNA 16-mers. Numbers correspond to target sites indicated in the sequence in (A). Ctr—control reaction without DNA oligonucleotide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556297&req=5

pone.0136395.g003: Induction of RNase H cleavage by potential helper oligomers.(A) Nucleotide sequence of the 5’UTRcvb3 RNA. Sites mapped as accessible to oligonucleotide hybridization are indicated with gray rectangles. Target sites of DNA 16-mers, potential helper oligomers, are marked with black lines below the sequence and numbered 1–19. (B) Representative agarose gels showing degradation of 5’UTRcvb3 RNA by RNase H in the presence of one of the DNA 16-mers. Numbers correspond to target sites indicated in the sequence in (A). Ctr—control reaction without DNA oligonucleotide.
Mentions: To design helper antisense oligomers which would be able to destabilize the double-stranded stems of the targeted RNA, we tested the binding efficiency of several DNA 16-mers complementary to the 5’UTRcvb3 using the RNase H assay. Nineteen DNA 16-mers were chosen for the test based on our previous mapping results and in several cases the oligomers were shifted in the 3’ direction by 2, 3 or 4 nucleotides (Fig 3A). It turned out that several oligonucleotides were capable of inducing efficient RNase H cleavage upon binding: these were oligomers Nos. 1, 2 and 4–9. The upper band on the gel corresponding to the full-length RNA was degraded in those cases (Fig 3B). The 16-mers most efficient at inducing RNase H activity, were oligomers Nos. 2, 4 and 5, complementary to domain II. Unexpectedly, three other oligomers Nos. 11, 14 and 18 were completely inactive in inducing RNA degradation, although they were directed into sites mapped previously as accessible to hybridization. Eight other oligomers Nos. 3, 10, 12, 13, 15–17, and 19, were also inefficient in inducing RNase H activity. These results highlight the need for testing oligomers of the final length for the ability to bind RNA regions mapped as accessible with short-oligomer-libraries. The sequences of all oligomers are shown in S2 Table.

Bottom Line: Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro.For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells.Addition of the helper oligomer improved silencing capacity of the respective siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.

No MeSH data available.


Related in: MedlinePlus