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Branched RNA: A New Architecture for RNA Interference.

Aviñó A, Ocampo SM, Perales JC, Eritja R - J Nucleic Acids (2011)

Bottom Line: These structures were used to obtain branched siRNA.Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition.As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Networking Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, 08028 Barcelona, Spain.

ABSTRACT
Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

No MeSH data available.


Schematic representation of the chemical structure of the branching units of the oligonucleotides described in this study.
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Related In: Results  -  Collection


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fig2: Schematic representation of the chemical structure of the branching units of the oligonucleotides described in this study.

Mentions: Figure 2 refers to the branched RNA structures synthesized in this study. DB stands for the symmetric doubler phosphoramidite obtained from commercial sources (Glen Research). Guanosine was protected with the dimethylaminomethylidene group, cytidine with the acetyl group, and adenosine with the benzoyl group. t-Butyldimethylsilyl (TBDMS) group was used for the protection of the 2′-OH function of the RNA monomers. The phosphoramidites were dissolved in dry acetonitrile (0.1 M), and a modified cycle was used with increased coupling time to 10 min. Oligoribonucleotide 1 was synthesized on a CPG solid support with a symmetric branching unit of two arms containing two DMT-protected hydroxyl groups, as described in [31]. Oligoribonucleotides 2 and 3 were synthesized using standard low-volume polystyrene thymidine columns. After the solid-phase synthesis, the supports were treated with concentrated aqueous ammonia-ethanol (3 : 1) for 1 h at 55°C. After filtration of the supports, the solutions were evaporated to dryness. The residue was dissolved in 85 μL of 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) for 12 h. Then, 85 μL of 1 M of triethylammonium acetate was added and the oligoribonucleotides were desalted on a NAP-10 column using water as eluent. The compounds were purified by HPLC under the following conditions. Column: Nucleosil 120–10 C18 (250  ×  4 mm); 20-min linear gradient from 15% to 100% B (DMT ON conditions); flow rate 3 mL/min; solution A was 5% acetonitrile in 0.1 M aqueous triethylammonium acetate (TEAA) buffer and B 70% acetonitrile in 0.1 M aqueous TEAA. The purified products were analyzed by MALDI-TOF mass spectrometry. Yields (0.2 μmol scale synthesis) were between 5–10 OD units at 260 nm.


Branched RNA: A New Architecture for RNA Interference.

Aviñó A, Ocampo SM, Perales JC, Eritja R - J Nucleic Acids (2011)

Schematic representation of the chemical structure of the branching units of the oligonucleotides described in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3065013&req=5

fig2: Schematic representation of the chemical structure of the branching units of the oligonucleotides described in this study.
Mentions: Figure 2 refers to the branched RNA structures synthesized in this study. DB stands for the symmetric doubler phosphoramidite obtained from commercial sources (Glen Research). Guanosine was protected with the dimethylaminomethylidene group, cytidine with the acetyl group, and adenosine with the benzoyl group. t-Butyldimethylsilyl (TBDMS) group was used for the protection of the 2′-OH function of the RNA monomers. The phosphoramidites were dissolved in dry acetonitrile (0.1 M), and a modified cycle was used with increased coupling time to 10 min. Oligoribonucleotide 1 was synthesized on a CPG solid support with a symmetric branching unit of two arms containing two DMT-protected hydroxyl groups, as described in [31]. Oligoribonucleotides 2 and 3 were synthesized using standard low-volume polystyrene thymidine columns. After the solid-phase synthesis, the supports were treated with concentrated aqueous ammonia-ethanol (3 : 1) for 1 h at 55°C. After filtration of the supports, the solutions were evaporated to dryness. The residue was dissolved in 85 μL of 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) for 12 h. Then, 85 μL of 1 M of triethylammonium acetate was added and the oligoribonucleotides were desalted on a NAP-10 column using water as eluent. The compounds were purified by HPLC under the following conditions. Column: Nucleosil 120–10 C18 (250  ×  4 mm); 20-min linear gradient from 15% to 100% B (DMT ON conditions); flow rate 3 mL/min; solution A was 5% acetonitrile in 0.1 M aqueous triethylammonium acetate (TEAA) buffer and B 70% acetonitrile in 0.1 M aqueous TEAA. The purified products were analyzed by MALDI-TOF mass spectrometry. Yields (0.2 μmol scale synthesis) were between 5–10 OD units at 260 nm.

Bottom Line: These structures were used to obtain branched siRNA.Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition.As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Networking Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, 08028 Barcelona, Spain.

ABSTRACT
Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

No MeSH data available.