Locked Nucleic Acid
LNA16/05.2005
By C. Lomholt
LNA Oligonucleotide Synthesis on an ABI3900 Instrument
The use of LNA phosphoramidites on an ABI3900 instrument follows standard procedures for use of phosphoramidites.
The LNA-A, G and T amidites are used as 0.05M solutions in anhydrous acetonitrile, in the case of LNA-mC it is advised to dissolve the amidite in a 25 tetrahydrofurane/acetonitrile1 solution (0.05M) to avoid precipitation of the amidite.
The best results are obtained when double coupling are used for LNA, a 2 x 75 seconds coupling time is found to be sufficient for all LNA phosphoramidites2.
LNA requires more oxidation compared to DNA. Triple oxidation is found to be optimal when synthesising standard LNA oligonucleotides.
| Molecular weight g/mole | Product Nr. | CAS. Nr. | Dissolve in | To obtain a 0.05M solution 100mg 250mg |
LNA-ABz | 885.9 | A-0063- | [206055-79-0] | Anhydrous Acetonitrile | 2.3 mL | 5.7 mL |
LNA-mCBz | 875.9 | mC-0066- | [206055-82-5] | THF/Acetonitrile 25/75 (v/v) | 2.3 mL | 5.7 mL |
LNA-GDMF | 852.9 | G-0082- | [709641-79-2] | Anhydrous Acetonitrile | 2.3 mL | 5.8 mL |
LNA-T | 772.8 | T-0064- | [206055-75-6] | Anhydrous Acetonitrile | 2.6 mL | 6.5 mL |
1 We recommend the use of anhydrous tetrahydrofurane (Aldrich 401757), as an alternative anhydrous dichloromethane (Aldrich 270997) can be used as a substitute for THF.
2 The consumption of LNA amidite is only 33% more compared to DNA since less amidite are used in each "single" coupling step.
Explanation to Synthesizer Cycle File:
When LNA amidites are used they are placed on the modifier positions 5, 6, 7, 8, 9 and 0. In the sequence the 5 to 0 positions are written in numbers, but to make modification in the synthesis cycle for these positions, except for the amidites, the modifications has to be made in the "Upper Case" cycle. Therefore it is advisable to use "Lower Case" for denoting DNA monomers in the sequence.
