Oligonucleotides, RNAi, DNAi purification
Oligonucleotides, often referred to as oligos, are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. Oligonucleotides are often used as probes for detecting complementary DNA or RNA because they bind readily to their complements.
Since 2001 and their description in mammalian cells, it has been reported that RNAi can induce sequence-specific silencing and can be used therapeutically in whole animals. In 2004 started the first phase I clinical trial of siRNA drug for age-related macular degeneration (AMD). Ever since, the interest toward RNAi has been exponentially growing. In 2006, Fire and Mello won Noble Prize in Physiology or Medicine for discovering RNAi mechanism.
DNA vaccine technology might offer an attractive approach to rapid manufacturing of efficient vaccines, since several human trials are under way with promising results. Efforts are being done to enhance purification of plasmid DNA, in order to achieve the best strategy providing an adequate product quality. The main challenge is the elimination of plasmid contaminants, where chromatography plays an important role.
AMBERCHROM™ XT20 is an excellent choice for the separation of synthetic oligonucleotides. A single step purification of a 20-mer synthetic, phosphodiester oligonucleotides was developed on a 10 (ID) x 250 mm Amberchrom profile XT20 column. The crude DMT-on oligonucleotides which was not fully deprotected, was loaded onto the column, detritylated and eluted. One of the advantages of AMBERCHROM™ XT20 is the ability to detrytilate on-column with the use of 0,5% trifluoroacetic acid (TFA).
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Column |
Amberchrom™ Profile™ XT20 (10x250) |
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Flow rate |
Loading and Equilibration: 4 ml/minute Detritylation: 4 ml/minute Elution: 1 ml/minute |
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Mobile Phase |
A: 0.1M TEAA, pH 7.0 B: 20% acetonitrile in 0.1M TEAA, pH 7.0 C: 0.5% TFA (for detritylation) |
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Gradient |
20% to 55% in 10 column volumes (200ml) | |
Sample |
20-mer phosphodiester oligonucleotide (5'- AGC TAG CTA GCT AGC TAG CT -3') DMT-on, not fully deprotected |
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Loading |
100 µl of 1.0 µmol | |
Detection |
UV @ 260 nm | |
Fractions |
1ml fractions collected | |
This example demonstrates the ability of AMBERCHROM™ media to purify crude synthetic oligonucleotides in a single step from a starting purity of ~46% to a final purity of ~91%.