Identification and Quantification of Protecting Groups Remaining in Commercial Oligonucleotide Products Using Monoclonal Antibodies

Fu, Chi Ling; Smith, Susanna; Simkins, Stephen G.; Agris, Paul F.

doi:10.1006/abio.2002.5687

Cited by 13 publications

(14 citation statements)

References 17 publications

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“…We often detect a mass addition of 53 or 106 Da, which represents one or two cyanoethyl functionalities, as well as other protection groups used in oligonucleotide synthesis (Fountain et al, 2003). Analyzing 25-110-mers by LC-MS, it was found that the incomplete postsynthesis deprotection commonly gives rise to additional peaks eluting after the target product (data not shown) (Fountain et al, 2003;Fu et al, 2002;Gilar, 2001).…”

Section: Lc-ms Analysis Of As-odnsmentioning

confidence: 95%

Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Gilár

Fountain²,

Budman³

et al. 2003

Oligonucleotides

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A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.

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Section: Lc-ms Analysis Of As-odnsmentioning

confidence: 95%

Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Gilár

Fountain²,

Budman³

et al. 2003

Oligonucleotides

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“…However, the analysis is insensitive because it depends on enzymatic cleavage of the oligonucleotide and on UV diode-array detection for identification and quantification. Capillary electrophoresis and mass spectrometry detect the aborted sequences, but are not easily adapted to identifying and quantifying the protecting groups that remain on the oligonucleotide 6 .…”

mentioning

confidence: 99%

“…To address this problem, we previously developed monoclonal antibodies (mAbs) for the specific identification and quantification of the nucleobase and sugar protecting groups commonly used in DNA and RNA chemical syntheses 6 . Using these mAbs, we now present a dot-blot assay and a microplate enzyme-linked immunosorbent assay (ELISA) for identification and quantification of protecting groups that remain in standard, intact DNA and RNA oligonucleotide samples (Fig.…”

mentioning

confidence: 99%

“…Thus, the mAb analysis is able to detect a single protected nucleoside in oligonucleotide samples containing 2 × 10 4 deprotected nucleosides. In contrast, HPLC nucleoside-composition analysis of enzyme-hydrolyzed DNA is limited to the detection of 2-5 nmol of protected nucleoside 6 . Using our present mAb dot-blot assay, 5 of 16 commercial DNA products obtained from eight different companies are found to have 1.0-5.2% contamination from benzoyl-and isopropylphenoxyacetyl-protecting groups ( Fig.…”

mentioning

confidence: 99%

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Erratum: The need for national centers for proteomics

Aebersold¹,

Watts²

2002

Nat Biotechnol

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“…Although decapping and deprotection are efficient reactions, the yield is not nearly 100%. Indeed, using monoclonal antibodies to specific protecting groups we previously showed that oligonucleotides from commercial sources claiming deprotection retained significant levels of protecting groups [see Refs ( 1 , 2 ); Barley-Maloney and Agris, in press]. This antibody-based method was previous validated using high-performance liquid chromatography (HPLC) ( 1 ).…”

Section: Introductionmentioning

confidence: 99%

Assessing incomplete deprotection of microarray oligonucleotides in situ

Dressman

Barley-Maloney

Rowlette

et al. 2006

Nucleic Acids Research

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En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

show abstract

Identification and Quantification of Protecting Groups Remaining in Commercial Oligonucleotide Products Using Monoclonal Antibodies

Cited by 13 publications

References 17 publications

Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Erratum: The need for national centers for proteomics

Assessing incomplete deprotection of microarray oligonucleotides in situ

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