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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

 

Solid phase synthesis of RNA oligonucleotides which are over 100-nt in length remains challenging due to the complexity of purification of the target strand from failure sequences. This work describes a non-chromatographic strategy that will enable routine solid phase synthesis of long RNA strands. 

Bond-breaking bio-orthogonal chemistry, consisting of a “click” reaction between trans-cyclooctene and tetrazine, followed by an intramolecular cyclization-driven uncaging step is described. The two-step process allows activation of caged compounds in biological media at neutral pH. The feasibility of this chemistry has been illustrated using NMR, while kinetics and pH-dependence were studied by fluorescence spectroscopy using caged coumarin. The practicality of the strategy is illustrated by activation of an anticancer drug, etoposide. 

A bio-orthogonal chemistry-based approach for fluorescent labelling of ribosomal RNA is described. It involves an adenosine analogue modified with trans -cyclooctene and masked 5′-phosphate group using aryl phosphoramidate. The incorporation into rRNA has been confirmed using agarose gel electrophoresis, as well as a highly sensitive UHPLC-MS/MS method. Fluorescent labelling of rRNA has been achieved in live HeLa cells via an inverse electron demand Diels–Alder reaction with a tetrazine conjugated to an Oregon Green fluorophore. This communication describes the stepwise approach that led to the development and characterization of the probe. The results demonstrate a new strategy towards development of future fluorescent probes to investigate the biochemistry of nucleic acids.