Promising oligo purification method wins more cash
(€589,079) by the US government to further develop its
oligonucleotide (oligo) purification technique which is
operationally similar to conventional reverse-phase (RP) methods
but offers better yields, selectivity and purity.
Synthetic pieces of DNA and RNA are used in numerous applications in genetics, diagnostics and therapeutics, with the total global market for oligos estimated to be worth around $500m and projected to grow to $776m by 2010.
But the production of oligos, based on solid-phase synthesis with hundreds of sequential chemical reactions, results in undesired by-products that must be removed by tedious purification techniques.
To develop more efficient separation methods, the US National Institute of General Medical Sciences (NIGMS) has awarded Berry & Associates a Phase II Small Business Innovation Research (SBIR) grant for their technique known as fluorous affinity purification.
Using an earlier Phase I SBIR grant, the company has already come up with fluorous tags, known as perfluoroalkyl 'ponytails', which are added to oligos using traditional solid-phase synthesis techniques and act as purification handles.
Thus, non-fluorous molecules which have not been tagged, likely to be failure sequences and other by-products, are rejected by a fluorinated adsorbent, much like Teflon releases all but other fluorinated substances.
Fluorous-tagged oligos, on the other hand, are strongly retained by the adsorbent, since fluorous-fluorous interactions are strong and selective, stronger than the hydrophobic interactions that are the basis of related methods involving RP adsorbent.
When the purified oligonucleotide is isolated, the fluorous tag is removed, allowing it to elute free of failure sequences, even when starting with long oligos.
Asked about scaling up this method, Will Pearson, the company's vice president for R&D told In-PharmaTechnologist.com this is viable, as it involves normal DNA/RNA synthesis techniques and RP-like purification methods.
"Of particular interest is the use of this technique as a 'first pass' purification for the volume production of oligonucleotide-based diagnostics and therapeutics with an initial fluorous purification removing failure sequences and non-oligonucleotide impurities in a highly selective fashion, affording a high recovery of enriched material that is ready for a final phase of purification, with ion-exchange chromatography for example," he said.
"On the other end of the spectrum of scale, fluorous affinity purification is ideal for the parallel purification of the small quantities of oligonucleotides obtained in high-throughput (HT) synthesis, especially with longer oligonucleotides, so 'catch and release' purification using solid-phase extraction (SPE) is an ideal match for HT methods."
With Phase II funding, Berry & Associates will expand the types of fluorous-tagged DNA/RNA building blocks that are available, allowing the purification of modified oligos such as optically labeled probes, phosphorylated oligonucleotides, and oligonucleotides with various functional groups such as amine and sulfhydryl.
"As with any new technique, there is always a barrier to getting scientists to try it," Pearson conceded.
"We believe that the strong affinity of fluorous-tagged oligonucleotides for fluorinated adsorbents will help our customers solve their purification problems, and we look forward to working with nucleic acids scientists in developing customised solutions."
Since the method does not require adopting new techniques, the company hopes its purification method will be welcomed by the industry.
