Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer.

The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer.

Denaturing reversed-phase HPLC using a mobile phase containing urea for oligodeoxynucleotide analysis.

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.

Purification of synthetic oligonucleotides via catching by polymerization.

This unit describes the purification of synthetic oligodeoxyribonucleotides (ODN) using a catching-by-polymerization approach. In a crude ODN, the major impurity is the failure sequences generated in the coupling step of each synthetic cycle. They are difficult to remove due to the similarity of their physical properties to the full-length sequences. Two non-chromatographic methods are described in the unit to solve the problem. In the first one, during automated synthesis, the failure sequences are tagged with a methacrylamide group, which is polymerizable and can participate in acrylamide radical polymerization reactions; the full-length sequences are not tagged. After synthesis, the crude mixture is subjected to polymerization. The failure sequences are incorporated into an insoluble polymer; the full-length sequences are extracted with water. In the second method, the full-length sequences are tagged with a methacrylamide group via a cleavable linker; the failure sequences are not tagged. After synthesis, the full-length sequences are incorporated into a polymer; the failure sequences are washed away with water. Pure full-length sequences are cleaved from the polymer. The two methods are complementary. .

Synthetic oligodeoxynucleotide purification by polymerization of failure sequences.

Synthetic oligodeoxynucleotide is purified by capping failure sequences with an acrylated phosphoramidite followed by polymerization and product extraction. The method is suitable for large scale oligonucleotide drug purification.

Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing, and releasing approach.

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides.