5′-DMT-protected double-stranded DNA: Synthesis and competence to enzymatic reactions
| Autor: | Andrei I. Zhauniarovich, Yuliya P. Burankova, Veronika V. Shchur, Sergei A. Usanov, Yaraslau V. Dzichenka, Aleksei V. Yantsevich |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
DNA Ligases
DNA polymerase Biophysics DNA Single-Stranded Polymerase cycling assembly 01 natural sciences Biochemistry 03 medical and health sciences chemistry.chemical_compound Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences DNA ligase DNA synthesis biology Oligonucleotide Chemistry 010401 analytical chemistry Cell Biology 0104 chemical sciences Exodeoxyribonucleases Polynucleotide biology.protein Nucleic acid DNA |
| Zdroj: | Analytical Biochemistry. 617:114115 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2021.114115 |
| Popis: | The functionalization of 5ʹ-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4ʹ-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5ʹ-DMT-protected ONs could allow producing both symmetric and asymmetric 5ʹ-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5ʹ-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5ʹ-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5ʹ-DMT modification of dsDNA does not affect activity of T5 5ʹ,3ʹ-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5ʹ-DMT-modification or search of ways to separate long 5ʹ-DMT-ssDNA and 5ʹ-OH-ssDNA could allow finding application of 5ʹ-DMT-modified oligo- and polynucleotides. |
| Databáze: | OpenAIRE |
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