Limits...
Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

Show MeSH

Related in: MedlinePlus

(A) Representative structure of the TuD RNAs. (B) Schematic representation of the generation of TuD RNA expression cassettes driven by mouse U6 promoter. 80–90mer synthetic oligonucleotides pairs are annealed and cloned between the two BsmBI sites present in the BamHI–EcoRI fragment (the original cassette) to generate TuD RNA expression cassettes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665227&req=5

Figure 2: (A) Representative structure of the TuD RNAs. (B) Schematic representation of the generation of TuD RNA expression cassettes driven by mouse U6 promoter. 80–90mer synthetic oligonucleotides pairs are annealed and cloned between the two BsmBI sites present in the BamHI–EcoRI fragment (the original cassette) to generate TuD RNA expression cassettes.

Mentions: From these results, we tentatively concluded that the structures of Decoy RNA #013 and #020 (Figure 2A) have great potential in the design of efficient inhibitors of miRNA function. These decoys contain two MBS regions, which are flanked by two stem structures through 3-nt-linkers. We have tentatively designated this decoy TuD (Tough Decoy) RNA and further analysed its characteristics and specificity as outlined below. The TuD RNA expression cassette can be quite readily constructed as shown in Figure 2B.Figure 2.


Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

(A) Representative structure of the TuD RNAs. (B) Schematic representation of the generation of TuD RNA expression cassettes driven by mouse U6 promoter. 80–90mer synthetic oligonucleotides pairs are annealed and cloned between the two BsmBI sites present in the BamHI–EcoRI fragment (the original cassette) to generate TuD RNA expression cassettes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665227&req=5

Figure 2: (A) Representative structure of the TuD RNAs. (B) Schematic representation of the generation of TuD RNA expression cassettes driven by mouse U6 promoter. 80–90mer synthetic oligonucleotides pairs are annealed and cloned between the two BsmBI sites present in the BamHI–EcoRI fragment (the original cassette) to generate TuD RNA expression cassettes.
Mentions: From these results, we tentatively concluded that the structures of Decoy RNA #013 and #020 (Figure 2A) have great potential in the design of efficient inhibitors of miRNA function. These decoys contain two MBS regions, which are flanked by two stem structures through 3-nt-linkers. We have tentatively designated this decoy TuD (Tough Decoy) RNA and further analysed its characteristics and specificity as outlined below. The TuD RNA expression cassette can be quite readily constructed as shown in Figure 2B.Figure 2.

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

Show MeSH
Related in: MedlinePlus