Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization

Pokharel, Durga; Yuan, Yinan; Fueangfung, Suntara; Fang, Shiyue

doi:10.1039/c3ra46986g

Cited by 8 publications

(18 citation statements)

References 27 publications

(24 reference statements)

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“…We found that 8-oxodG could be detected by HPLC. In contrast, 8-oxo-dG could not be detected from nucleosides from digesting 2 [14]. In addition, we had subjected the G nucleoside into the radical polymerization conditions and found that it was stable [16].…”

Section: Introductionmentioning

confidence: 99%

“…For example, to reduce costs, in some of our previous experiments, phosphoramidite solutions that were stored in a freezer under nitrogen in jars containing Drierite over half a year were used. The ODNs purified by catching by ploymerization were found contaminated with small amount of unidentifiable impurities [14]. After realizing this, we always used phosphoramidite solutions prepared within one week and found that ODN purity was consistently 100% after purification by polymerization.…”

Section: Introductionmentioning

confidence: 99%

“…We recently reported a catching failure sequences by polymerization method for ODN purification [13,14]. In this method, the failure sequences are capped with a poly merizable phosphoramidite such as 1 (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…The ODN synthesis procedure, which includes capping failure sequences by 1, was similar as previously described [14]. Briefly, the synthesizer manufacturer (ABI 394) suggested 1 µmol DNA synthesis cycle was copied to create a new cycle.…”

Section: Introductionmentioning

confidence: 99%

“…Following capping, three washes (acetonitrile to column, 10 seconds; reverse flush, 6 seconds) were added. The synthesis was set up as usual with 1 (0.1 M in acetonitrile) [14] being placed in bottle 5. 4,5-Dicyanoimidazole (0.25 M in acetonitrile) was used as activator.…”

Section: Introductionmentioning

confidence: 99%

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A Highly Convenient Procedure for Oligodeoxynucleotide Purification

Pokharel¹,

Fang²

2014

TOOCJ

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Purification of synthetic oligodeoxynucleotides (ODNs) is simply achieved by capping failure sequences with a polymerizable phosphoramidite followed by polymerization. In this article, the reduction of the amount of polymerization monomer to drastically increase ODN extraction efficiency, the use of a centrifugal filterunit to ease the extraction process and the notification of using fresh phosphoramidite solutions are described. In addition, further evidence to support the purity of ODN and discussions of ODN stability under radical polymerization conditions are provided.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A Highly Convenient Procedure for Oligodeoxynucleotide Purification

Pokharel¹,

Fang²

2014

TOOCJ

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Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis

Fang

Eriyagama

Yuan

et al. 2020

CP Nucleic Acid Chemistry

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This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-ylmethoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides, and α-chloroamides-which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies-can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups.

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A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences

Grajkowski

Cieślak

Beaucage

2017

CP Nucleic Acid Chemistry

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An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences.

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Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization

Abstract: Synthetic oligodeoxynucleotides are simply purified by capping failure sequences with a methacrylamide phosphoramidite, co-polymerization with N,N-dimethylacrylamide and extraction with water.

Cited by 8 publications

References 27 publications

A Highly Convenient Procedure for Oligodeoxynucleotide Purification

A Highly Convenient Procedure for Oligodeoxynucleotide Purification

Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis

A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences

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