Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides
| Autor: | Kacper Skakuj, Katherine E. Bujold, Chad A. Mirkin |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Gel electrophoresis
0303 health sciences Nuclease biology Oligonucleotide Thiophenol 030305 genetics & heredity Oligonucleotides General Medicine Combinatorial chemistry Article 03 medical and health sciences chemistry.chemical_compound Automation Monomer chemistry Thiourea biology.protein Nucleic Acid Conformation Guanidine DNA 030304 developmental biology |
| Zdroj: | Curr Protoc Nucleic Acid Chem |
| Popis: | This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer. |
| Databáze: | OpenAIRE |
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