Sequence verification of oligonucleotides containing multiple arylamine modifications by enzymatic digestion and liquid chromatography mass spectrometry (LC/MS)
| Autor: | Lan Gao, Li Zhang, M. Paul Chiarelli, Bongsup P. Cho |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Exonucleases
Exonuclease Spectrometry Mass Electrospray Ionization Electrospray ionization Oligonucleotides Article Isomerism Structural Biology Liquid chromatography–mass spectrometry Enzymatic hydrolysis Cleave Escherichia coli Nucleotide Amines Spectroscopy chemistry.chemical_classification Chromatography biology Oligonucleotide Hydrolysis Nucleic acid sequence hemic and immune systems Sequence Analysis DNA respiratory system Endonucleases chemistry biology.protein Spectrophotometry Ultraviolet Chromatography Liquid |
| Zdroj: | Journal of the American Society for Mass Spectrometry. 19:1147-1155 |
| ISSN: | 1044-0305 |
| Popis: | An analytical method for the structure differentiation of arylamine modified oligonucleotides (ODNs) using on-line LC/MS analysis of raw exonuclease digests is described. Six different dodeca ODNs derived from the reaction of N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene with the dodeca oligonucleotide 5'-CTCGGCGCCATC-3' are isolated and sequenced with this LC/MS method using 3'- and 5'-exonucleases. When the three products modified by a single aminofluorene (AF) are subjected to 3'-exonuclease digestion, the exonuclease will cleave a modified nucleotide but when di-AF modified ODNs are analyzed the 3'-exonuclease ceases to cleave nucleotides when the first modification is exposed at the 3'-terminus. Small abundances of ODN fragments formed by the cleavage of an AF-modified nucleotide were observed when two of the three di-AF modified ODNs were subjected to 5'-exonuclease digestion. The results of the 5'-exonuclease studies of the three di-AF modified ODNs suggest that as the number of unmodified bases between two modifications in an ODN sequence increases, the easier it becomes to sequence beyond the modification closest to the 5'-terminus. The results of this study indicate that the LC/MS method described here would be useful in sequencing ODNs modified by multiple arylamines to be used as templates for site-specific mutagenesis studies. |
| Databáze: | OpenAIRE |
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