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Abstract This protocol describes the synthesis of long oligonucleotides (up to 401‐mer), their isolation from complex mixtures using the catching‐by‐polymerization (CBP) method, and the selection of error‐free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid‐phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full‐length sequence with a polymerizable tagging phosphoramidite (PTP), co‐polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full‐length sequence from the polymer. Cloning and sequencing guided selection of error‐free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher‐order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Long oligo synthesis Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP) Support Protocol 2: Synthesis of 5′‐O‐Bz phosphoramidite Basic Protocol 2: Catching‐by‐polymerization (CBP) purification Basic Protocol 3: Error‐free sequence selection via cloning and sequencing
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Synthesis of 5‐Cyanomethyluridine (cnm 5 U) and 5‐Cyanouridine (cn 5 U) Phosphoramidites and Their Incorporation into RNA Oligonucleotides
Abstract This article contains detailed synthetic protocols for preparation of 5‐cyanomethyluridine (cnm5U) and 5‐cyanouridine (cn5U) phosphoramidites. The synthesis of the cnm5U phosphoramidite building block starts with commercially available 5‐methyluridine (m5C), followed by bromination of the 5‐methyl group to install the cyano moiety using TMSCN/TBAF. The cn5U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5‐cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA‐based therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cnm5U and cn5U phosphoramidites Basic Protocol 2: Synthesis, purification, and characterization of cnm5U‐ and cn5U‐modified RNA oligonucleotides
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- Award ID(s):
- 1715234
- PAR ID:
- 10238704
- 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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