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Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates
A A Padmapriya1, J Tang, S Agrawal
1Hybridon, Inc., Worcester, Massachusetts 01605.
Antisense Research and Development
|January 1, 1994
Summary
Automated DNA synthesis enables efficient production of long-chain oligodeoxyribonucleotides on millimole scales. Optimized protocols yield high-purity oligonucleotides suitable for various applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Organic Chemistry
Background:
- Automated DNA synthesis is crucial for producing oligonucleotides.
- Scaling up oligonucleotide synthesis presents challenges in efficiency and purity.
Purpose of the Study:
- To optimize automated DNA synthesis protocols for millimole scales.
- To achieve high coupling efficiency and purity in synthesized oligonucleotides.
Main Methods:
- Utilized a modified automated DNA synthesizer for 1-, 2-, and 5-mmol scale synthesis.
- Optimized reaction cycles for efficient coupling (> or = 97%).
- Purified oligonucleotides using reversed-phase liquid chromatography, detritylation, and desalting.
Main Results:
- Successfully synthesized a 25-mer oligodeoxyribonucleotide on millimole scales.
- Achieved approximately 2.4, 4.8, and 12 g of purified product at 1, 2, and 5 mmol scales, respectively.
- Characterized purified oligonucleotides using 31P NMR, mass spectrometry, capillary gel electrophoresis, and ion-exchange HPLC.
Conclusions:
- Developed and validated scalable protocols for automated oligonucleotide synthesis.
- Demonstrated the feasibility of producing significant quantities of high-purity oligonucleotides.
- The optimized methods provide a reliable approach for large-scale oligonucleotide production.