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The catching-by-polymerization (CBP) oligodeoxynucleotide (oligo or ODN) purification method has been demonstrated suitable for large-scale, parallel, and long oligo purification. The authenticity of the oligos has been verifiedviaDNA sequencing, and gene construction and expression. A remaining obstacle to the practical utility of the CBP method is affordable polymerizable tagging phosphoramidites (PTPs) that are needed for the method. In this article, we report scalable synthesis of the four nucleoside (dA, dC, dG and T) precursors to the PTPs using a route having five steps from inexpensive starting materials. The overall yields ranged from 21% to 35%. The scales of the synthesis presented here are up to 2.1 grams of the precursors. Because the syntheses are chromatography-free, further scaling up the syntheses of the precursors have become more feasible. With the precursors, the PTPs can be synthesized in one step using standard methods involving a chromatography purification.
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De Novo Synthesis of Error‐Free Long Oligos
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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- PAR ID:
- 10559352
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 4
- Issue:
- 10
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile‐sensitive epigeneticN4‐acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α‐haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3‐dithian‐2‐yl‐methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo‐amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non‐nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc‐CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid‐protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc‐CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc‐CE phosphoramiditemore » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/cpz1.70028
