Fluorous Affinity Purification of Oligonucleotides
Fluoro-Pak™ Columns and Fluorous Phosphoramidites
Fluorous Affinity Purification of Oligonucleotides is a quick and simple affinity-based method for the purification of oligonucleotides that relies on the strong interaction of fluorous-tagged oligonucleotides with the fluorinated adsorbent present in Fluoro-Pak™ columns. Fluorous affinity purification is operationally similar to DMT-on purification using a reverse-phase (RP) adsorbent, e.g. RP cartridge purification. Jump to fluorous products and pricing.
The fluorous affinity purification method affords the following advantages over traditional DMT-on RP purification:
- One-pass loading without ammonia removal.
- High selectivity for removal of failure sequences, even with long oligonucleotides. Fluorous-tagged oligonucleotides are strongly retained, whereas non-fluorous materials (failure sequences, other by-products) are poorly retained.
- High recoveries (typically 70-100%), free of failure sequences.
- Excellent for longer oligonucleotides (e.g. 50-100+ mers), where recoveries are nearly quantitative.
- Does not require adopting new techniques.
What does "fluorous" mean, and what is the nature of the fluorous affinity interaction? Highly fluorinated organic compounds are both hydrophobic and lipophobic, preferring instead to associate with other fluorinated substances. Organic molecules that have both an organic domain and a perfluoroalkyl domain (e.g., a linear perfluoroalkyl "ponytail") are known as fluorous molecules, (not to be confused with fluorescent molecules!)and may be separated from nonfluorous molecules by interaction with fluorinated separation media such as Fluoro-Pak columns. Fluorous-fluorous interactions are strong and selective ("like dissolves like"). Regardless of the structure of the oligonucleotide, the fluorous tag dominates, consistently moving such molecules to longer retention times and therefor away from non-fluorous-tagged by-products. The fluorous tail "wags the dog".
How is the fluorous method used for oligonucleotide purification? Fluorous Affinity Purification of Oligonucleotides is similar to DMT-on RP cartridge purification, and we have developed protocols that are as "plug-and-play" as possible. The first step is to install a single nucleotide at the 5'-terminus of the oligonucleotide using a fluorous-tagged phosphoramidite. As shown below, the fluorous tag can take the form of a fluorous dimethoxytrityl ("FDMT") group or a fluorous monomethoxytrityl ("FMMT") group, where a fluorous ponytail is attached via an ethylene spacer to a normal DMT or MMT group. The fluorous trityl groups are designed so that they behave just like the non-fluorous versions: the rates of detritylation and absorption maxima of the trityl cations are very similar to what is observed for the parent compounds. Please note that only one coupling of a fluorous-trityl phosphoramidite is required; normal DMT amidites are used for the earlier steps in the synthesis. The synthesis is run in DMT-on mode, leaving the FDMT or FMMT group in place. FDMT- and FMMT-bearing amidites are entirely soluble in acetonitrile and couple normally.
The FDMT- and FMMT-on oligonucleotides are cleaved from the solid support as usual and the base protecting groups are removed according to standard methods. If ammonia is used, it is not necessary to evaporate it. The crude deprotection solution is diluted with a salt-containing loading buffer and applied to a Fluoro-Pak™ column. Binding of the fluorous-tagged oligonucleotide occurs in one pass, leaving most of the failure sequences unbound. Washing with 5-10% acetonitrile in 0.1 M TEAA removes the rest of the failures. On-column detritylation with TFA followed by elution of the purified oligonucleotide is then carried out. Alternatively, the fluorous trityl oligonucleotide can be eluted from the column and detritylated in a separate operation.
Example: Fluorous-tagged mixed-base 75-mers (200 nmol scale) were purified by Fluorous Affinity Purification to provide 9-11 A(260) units of the fully deprotected material. This represents a nearly quantitative recovery of the available FDMT-tagged oligonucleotides present in the crude ammonia deblock solutions (estimated by HPLC). The final 75-mers were analyzed by RP-HPLC (see trace below).
What can the fluorous reagents be used for?
- Installation of removable fluorous tags into oligonucleotides.
- Installation of a permanent fluorous tag at any position to enable spotting on fluorous-coated glass slides.
- Simultaneous 5'-phosphorylation and fluorous affinity purification.
- Enable FDMT-on fluorous affinity purification without purchasing separate FDMT-bearing phosphoramidites.
- Simultaneous introduction of a fluorous tag and a fluorescein label.