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Abstract Solid‐phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non‐chromatographic procedure that will enable routine solid‐phase synthesis and purification of long RNA strands. The optimized five‐step process is based on bio‐orthogonal inverse electron demand Diels‐Alder chemistry betweentrans‐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 while on‐support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76‐nt long tRNA and 101‐nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR‐Cas9 experiments. © 2021 Wiley Periodicals LLC. Basic Protocol: Five‐step non‐chromatographic purification of synthetic RNA oligonucleotides Support Protocol 1: Synthesis of the components that are required for the non‐chromatographic purification of long RNA oligonucleotides. Support Protocol 2: Solid‐phase RNA synthesis
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Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry
Abstract This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry
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- PAR ID:
- 10185509
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Nucleic Acid Chemistry
- Volume:
- 82
- Issue:
- 1
- ISSN:
- 1934-9270
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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