Sequence Verification of Oligonucleotides Containing Multiple Arylamine Modifications by Enzymatic Digestion and Liquid Chromatography Mass Spectrometry (LC/MS)

• Published in: Journal of the American Society for Mass Spectrometry Vol. 19; no. 8; pp. 1147 - 1155
• Main Authors: Gao, Lan, Zhang, Li, Cho, Bongsup P., Chiarelli, M. Paul
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01-08-2008; Springer-Verlag; Elsevier Science; Springer Nature B.V
• Subjects: Amines; Amines - chemistry; Analytical Chemistry; Analytical, structural and metabolic biochemistry; Bioinformatics; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid; Digestion; Endonucleases - chemistry; Escherichia coli - chemistry; Exact sciences and technology; Exonucleases - chemistry; Fragments; Fundamental and applied biological sciences. Psychology; Hydrolysis; Isomerism; Liquid chromatography; Mass spectrometry; Nucleic acids; Nucleic bases, nucleotides; Nucleotides; Oligonucleotides; Oligonucleotides - analysis; Organic Chemistry; Organic compounds; Other chromatographic methods; Proteomics; Sequence Analysis, DNA; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Aminofluorene; Small Abundance; Endonuclease Activity; Guanine; Trifluoroacetyl; Nucleotide sequence; Coupled method; Time of flight method; Analysis; Enzymatic hydrolysis; HPLC chromatography; Oligonucleotide; Arylamine; Fluorene derivatives; Mass spectrometry; Aromatic amine
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2008.04.034

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.