TY - JOUR
T1 - Locating nucleobase lesions within DNA sequences by MALDI-TOF mass spectral analysis of exonuclease ladders
AU - Tretyakova, Natalia Y
AU - Matter, B.
AU - Ogdie, A.
AU - Wishnok, J. S.
AU - Tannenbaum, S. R.
PY - 2001
Y1 - 2001
N2 - The location of carcinogen-modified nucleobases (DNA adducts) within DNA sequences is a critical factor affecting their promutagenic properties and persistence in DNA. We now report the use of controlled exonuclease digestion followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly map modified nucleobases within DNA. The DNA sequence is determined by mass spectral analysis of the DNA ladders produced by sequential removal of nucleotides with either 5′→3′ or 3′→5′ exonuclease. Individual mononucleotides are identified from the mass differences between adjacent peaks corresponding to singly charged ions of the products of enzymatic cleavage. Chemically modified nucleotides are detected and identified by their molecular weight. The resolution and mass accuracy of this approach are sufficient to identify nucleobase modifications differing in mass by as little as 2 Da. No a priori information on the DNA sequence or adduct type is required. We demonstrate the general applicability of this method by sequencing synthetic oligonucleotides containing a range of nucleobase modifications: O6-methylguanine, peroxynitrite-induced oxidative lesions (oxaluric acid, oxazolone, cyanuric acid), and the N2-guanine adduct of (+,-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydribenzo[a]pyrene. Sequence information is also obtained for DNA oligodeoxynucleotides containing O6-pyridyloxobutylguanine, despite the ability of this lesion to block 3′-phosphodiesterase.
AB - The location of carcinogen-modified nucleobases (DNA adducts) within DNA sequences is a critical factor affecting their promutagenic properties and persistence in DNA. We now report the use of controlled exonuclease digestion followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly map modified nucleobases within DNA. The DNA sequence is determined by mass spectral analysis of the DNA ladders produced by sequential removal of nucleotides with either 5′→3′ or 3′→5′ exonuclease. Individual mononucleotides are identified from the mass differences between adjacent peaks corresponding to singly charged ions of the products of enzymatic cleavage. Chemically modified nucleotides are detected and identified by their molecular weight. The resolution and mass accuracy of this approach are sufficient to identify nucleobase modifications differing in mass by as little as 2 Da. No a priori information on the DNA sequence or adduct type is required. We demonstrate the general applicability of this method by sequencing synthetic oligonucleotides containing a range of nucleobase modifications: O6-methylguanine, peroxynitrite-induced oxidative lesions (oxaluric acid, oxazolone, cyanuric acid), and the N2-guanine adduct of (+,-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydribenzo[a]pyrene. Sequence information is also obtained for DNA oligodeoxynucleotides containing O6-pyridyloxobutylguanine, despite the ability of this lesion to block 3′-phosphodiesterase.
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U2 - 10.1021/tx010062i
DO - 10.1021/tx010062i
M3 - Article
C2 - 11511180
AN - SCOPUS:0034860461
SN - 0893-228X
VL - 14
SP - 1058
EP - 1070
JO - Chemical research in toxicology
JF - Chemical research in toxicology
IS - 8
ER -