Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection.
We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.
Cornelius
Michael
M
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Wörth
Christian G C T
CG
Kliem
Hans-Christian
HC
Wiessler
Manfred
M
Schmeiser
Heinz H
HH
D001896:Boron Compounds / Q000737:chemistry*; D004247:DNA / Q000187:drug effects; D003838:Deoxyadenine Nucleotides / Q000302:isolation & purification; D003843:Deoxycytidine Monophosphate / Q000302:isolation & purification; D003848:Deoxyguanine Nucleotides / Q000302:isolation & purification; D003854:Deoxyribonucleotides / Q000032:analysis* / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005029:Ethylenediamines / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007834:Lasers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013447:Sulfites / Q000737:chemistry; D013938:Thymidine Monophosphate / Q000302:isolation & purification
C503730:4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine; D001896:Boron Compounds; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D013447:Sulfites; D003843:Deoxycytidine Monophosphate; D013938:Thymidine Monophosphate; C012873:2'-deoxy-5'-adenosine monophosphate; C007257:2'-deoxyguanosine 5'-phosphate; D004247:DNA; C042345:hydrogen sulfite