A new mass spectrometric approach to detect modifications in DNA

Abstract

A new approach is described for the enzymatic digestion of DNA yielding oligonucleotides ranging from dinucleoside monophosphates to octanucleoside heptaphosphates. DNA was digested by means of the benzon nuclease, an unspecific nuclease, and alkaline phosphatase to remove the terminal phosphate. The mixture of oligonucleotides was separated using capillary-zone electrophoresis with a buffer system, yielding a rather strong electro-osmotic flow. The oligomers are separated into groups with nucleotides of the same chain length. The separation capillary was used as the innermost capillary of an electrospray spraying system. Negative molecular ions of the nucleotides were recorded using a home-built interface and ion source for a sector-field mass spectrometer. This approach allows the facile detection of DNA modifications since they lead not only to differences in mass, but also can possess altered electrophoretic mobility. For modifying reactions which exhibit sequence specificity, the information is retained in the oligomers. Thus, reactions of DNA with electrophiles can be evaluated at different levels, since in longer chains, even complex sequence specificity may be apparent. Results from calf thymus DNA digests and preliminary experiments with DNA adducts with styrene oxide are discussed.

Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

“…The presence of residual DNA in the final viral preparations, derived from host producer cells and plasmid contamination (Chen et al, 2001), should be reduced in their amount and size to below approximately 200 base pairs in order to avoid the transfer of a residual functional open reading frame (FDA Guidance for Industry, 2010) and to further increase the product's purity. DNAses, such as Benzonase endonuclease, are often used to decrease the DNA load (Sastry et al, 2004) and to reduce the length of oligonucleotide fragments down to 3-8 bp ( Janning et al, 1994). In this work, purified and nonpurified samples (after and before AEXc, respectively) were incubated with 50 U/ml of Benzonase for 30 min at 37°C.…”

Recent developments in the determination of urinary cancer biomarkers by capillary electrophoresis

“…Other than quantitative improvement, MS can also provide detailed structural information using collision-induced dissociation (CID) or post-source decay (PSD) [70]. Since CE-ESI-MS was first applied for analysis of DNA adducts in 1994 by Janning et al [75], numerous studies have been conducted to detect modified oligonucleotides and modified nucleotides. The researches that have performed on determination of modified oligonucleotides included styrene oxide-modified oligonucleotides [75,82], alkylated oligonucleotides and oligonucleotides containing halogenated nucleobases [79], N-(acetylamino) fluorine-modified oligonucleotides [83], and unmodified oligonucleotides [77].…”

“…Since CE-ESI-MS was first applied for analysis of DNA adducts in 1994 by Janning et al [75], numerous studies have been conducted to detect modified oligonucleotides and modified nucleotides. The researches that have performed on determination of modified oligonucleotides included styrene oxide-modified oligonucleotides [75,82], alkylated oligonucleotides and oligonucleotides containing halogenated nucleobases [79], N-(acetylamino) fluorine-modified oligonucleotides [83], and unmodified oligonucleotides [77]. The modified nucleotides that have been quantitatively studied included N 7 , O-6, N 2 -modified guanosine monophosphate [82],…”

Analysis of nucleic acid constituents by on‐line capillary electrophoresis‐mass spectrometry

“…The first application of CZE coupled to ESI-MS for the analysis of DNA adducts was demonstrated by Janning et al [76]. Styrene oxide-modified DNA was subjected to partial enzymatic digestion by benzon nuclease yielding a complex mixture of modified and unmodified oligonucleotides ranging from di-to octanucleotides.…”