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0. 10. 20. 30. 40. 50. 60. Time [min.]. Synthetic Oligodeoxynucleotide Purification by Polymerization of Failure Sequences Suntara Fueangfung 1 , Xi lin 1 , and SHIYUE FANG 1 1 Department of Chemistry, Michigan Technological University. Introduction .

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Synthetic Oligodeoxynucleotide Purification by Polymerization of Failure Sequences

Suntara Fueangfung1, Xi lin1,and SHIYUE FANG1

1Department of Chemistry, Michigan Technological University

Introduction

For a typical synthesis of a 20-mer ODN on a controlled pore glass (CPG), the failure sequences constitute 30-60% of the total ODN content. Because these impurities have the same physical properties as the full-length ODN, they are difficult to remove. The polyacrylamide gel electrophoresis (PAGE) can efficiently separate them, but this method cannot be scaled up. HPLC methods, especially trityl-on reverse phase (RP) and anion exchange HPLC, have been adapted to purify large scale ODNs, which removes impurities including failure sequences. However these methods require expensive instruments and preparative columns. In addition , large volumes of solvent are needed, which must be subsequently evaporated. As a result, the method is very expensive for large scale purification.

In this poster, we report a novel synthetic oligodeoxynucleotide purification method by polymerization of failure sequences, which only requires cheap reagents and simple operations.

HPLC Traces and Results

In recent years, recognition of the ability of oligodeoxynucleotides (ODNs) to selectively silence gene expression , and thus potentially treat a wide range of human diseases, demands quantities of pure ODNs at the kilogram to metric ton scales. Synthesis at this level is now possible using solid phase synthesis and phosphoramidite chemistry (see Figure 1). Besides the desired full-length sequence, the crude ODN contains impurities, which include truncated failure sequences generated in each synthetic cycle due to incomplete coupling, small molecules resulting from protecting groups, and minute quantities of deletion sequences from incomplete detritylation and incomplete capping, and addition sequences from premature detritylation before and during coupling.

a

Crude ODN

ODN after polymerization

b

c

ODN after polymerization and desalting

ODN after polymerization,

desalting and NH3 treatment

d

e

Pure ODN +control

Proposed Purification Method of ODNs

Control ODN

f

Instead of capping with acetic anhydride, the failure sequences would be capped with methacrylatedphosphoramidite1 (Figure 2). Followed by radical acrylamide polymerization, the failure sequences polymerized and leave full-length ODNs in aqueous phase. With water extraction, full-length ODNs could be retrieved.

g

ODN after polymerization,

NH3 and BuOH treatment

Figure 3. Reverse phase HPLC traces of ODN 2 purification process: (a) crude; (b) after polymerization; (c) after size exclusion chromatography; (d) after treating with NH4OH; (e) co-injection with authentic ODN; (f) authentic ODN; (g) purified by polymerization and n-BuOH precipitation.

Pure ODN was obtained after polymerization and n-BuOHprecipitation and the yield for purification process was estimated to be 83 %.

Full-length ODN was confirmed by co-injection with authentic ODN and MALDI-TOF analysis (calculated mass for [M–H]⎯: 6057, found mass for [M–H]⎯: 6057).

Experimental Scheme

Conclusion

We have developed a new method for ODN purification. This method does not need any chromatography or expensive reagents and purification is achieved by simple operation such as shaking and extraction. Therefore, it is suitable for purification of large scale of ODN drugs.

Acknowledgement

MTU Chemistry Department and Biotech Research Center;

Michigan Universities Commercialization Initiative;

National Science Foundation