Standard DNA phosphoramidites,
solid supports, and reagents were purchased from Link Technologies
and Applied Biosystems. LNA phosphoramidites were obtained from Exiqon.
Automated solid-phase synthesis of oligonucleotides (trityl off) was
performed on an Applied Biosystems 394 synthesizer. The synthesis
was performed on 1.0 μm scale involving cycles of acid-catalyzed
detritylation, coupling, capping, and iodine oxidation. Standard DNA
phosphoramidites were coupled for 60 s, whereas extended coupling
time of 10 min was used for the modified phosphoramidites including
LNA phosphoramidites. Modified phosphoramidites12 , 13 , 14 , and 15 were used to prepare
ONs with monomersW , X , Y ,
andZ respectively. Coupling efficiencies and overall
synthesis yields were determined by the inbuilt automated trityl cation
conductivity monitoring facility and were ≥98.0% in all the
cases. The oligonucleotides were then cleaved from the solid support
and protecting groups from the nucleobase and backbone were removed
by exposure to concentrated aqueous ammonium hydroxide for 60 min
at room temperature followed by heating in a sealed tube for 5 h at
55 °C. RNA was purchased from Integrated DNA Technologies BVBA,
Leuven, Belgium.
solid supports, and reagents were purchased from Link Technologies
and Applied Biosystems. LNA phosphoramidites were obtained from Exiqon.
Automated solid-phase synthesis of oligonucleotides (trityl off) was
performed on an Applied Biosystems 394 synthesizer. The synthesis
was performed on 1.0 μm scale involving cycles of acid-catalyzed
detritylation, coupling, capping, and iodine oxidation. Standard DNA
phosphoramidites were coupled for 60 s, whereas extended coupling
time of 10 min was used for the modified phosphoramidites including
LNA phosphoramidites. Modified phosphoramidites
ONs with monomers
and
synthesis yields were determined by the inbuilt automated trityl cation
conductivity monitoring facility and were ≥98.0% in all the
cases. The oligonucleotides were then cleaved from the solid support
and protecting groups from the nucleobase and backbone were removed
by exposure to concentrated aqueous ammonium hydroxide for 60 min
at room temperature followed by heating in a sealed tube for 5 h at
55 °C. RNA was purchased from Integrated DNA Technologies BVBA,
Leuven, Belgium.