Locked nucleic acid synthesis.
Methods and protocols for automated synthesis and purification of locked nucleic acid (LNA), a class of oligonucleotides obeying the Watson-Crick base-pairing rules but displaying unprecedented binding affinities toward complementary deoxyribonucleic acid (cDNA) and ribonucleic acid (RNA), is described. LNA and LNA-DNA chimeras containing phosphordiester or phosphorothioate linkages, or a mixture thereof, can be assembled by standard DNA synthesizers using 2-cyanoethyl DNA phosphoramidites and 2-cyanoethyl LNA phosphoramidites. Compared to the standard protocols used for DNA synthesis, slightly longer coupling time and oxidation time are needed for efficient oligomerization of LNA phosphoramidites. When the LNA has been assembled, it is removed from the solid support as the 5'-end-O-dimethoxytrityl (DMT) protected LNA oligomer by treatment with concentrated aqueous ammonia that also removes the phosphate and nucleobase protecting groups. The crude DMT protected LNA product can be purified using, for example, reversed-phase chromatography.
Pfundheller
Henrik M
HM
Department of Chemistry, Exiqon A/S, Vedbaek, Denmark.
Sørensen
Anders M
AM
Lomholt
Christian
C
Johansen
Anders M
AM
Koch
Troels
T
Wengel
Jesper
J
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D009696:Nucleic Acids / Q000138:chemical synthesis*; D021241:Spectrometry, Mass, Electrospray Ionization
D009696:Nucleic Acids