| Autor: |
Jia Sheng, Maksim Royzen, Muhan He, Phensinee Haruehanroengra, Xunshen Wu, Irfan Khan |
| Rok vydání: |
2020 |
| Předmět: |
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| DOI: |
10.1101/2020.10.09.334060 |
| Popis: |
Solid phase synthesis of RNA oligonucleotides which are over 100-nt in length remains to be challenging due to the complexity of purification of the target strands from the failure sequences. This work describes a non-chromatographic strategy that will enable routine solid phase synthesis of long RNA strands. The optimized five-step process is based on bio-orthogonal inverse electron demand Diels-Alder chemistry between trans-cyclooctene (TCO) and tetrazine (Tz) and entails solid phase synthesis of RNA on a photo-labile support. The target oligonucleotide strands are selectively tagged with Tz. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach was optimized using a model 20-mer DNA strand and was successfully applied towards synthesis of 76-nt long tRNA and 101-nt long sgRNA. Purity of the isolated oligonucleotides was evaluated using gel electrophoresis and mass spectrometry, while functional fidelity of the sgRNA was confirmed using CRISPR-Cas9 experiments and flow cytometry. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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