Staphylococcal nuclease high-performance liquid chromatographic characterization of diaminooctane-modified DNA and its biotin and fluorescein derivatives.
DNA was subjected to bisulfite-catalyzed transamination at the N4 sites of its cytosine residues with 1,8-diaminooctane (DAO). The product, DNA-DAO, was non-specifically degraded with a cloned staphylococcal nuclease (Nase). The products from the Nase digestion were determined by high-performance liquid chromatography (HPLC) to define the extent of reaction with DAO. Mostly, nucleoside 3'-monophosphates were obtained, along with four Nase-resistant dinucleotides: TpdGp, dApdGp, TpdCp-DAO and dApdCp-DAO. The addition of spleen phosphodiesterase II gave a faster hydrolysis and left no dinucleotides. A mixture of Nase, snake venom phosphodiesterase I and alkaline phosphatase gave a fast hydrolysis as well but two dinucleotides, apparently TpdC-DAO and dApdC-DAO, persisted. Further modification of the diaminooctyl side chains with fluorescein isothiocyanate or biotin N-hydroxysuccinimide ester was similarly investigated. Interestingly, derivatization of the DAO side chain with biotin eliminates the resistance of TpdCp-DAO and dApdCp-DAO to Nase digestion. This work provides some guidelines for using Nase, alone or with other nucleases, along with HPLC, for characterizing alkyldiamine DNA products, and should be similarly useful for studying other modifications of DNA.
Cecchini
D J
DJ
Department of Medicinal Chemistry, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115.
Guan
K L
KL
Giese
R W
RW
D000469:Alkaline Phosphatase / Q000032:analysis; D001710:Biotin; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D003959:Diamines; D019793:Fluorescein; D005452:Fluoresceins; D007202:Indicators and Reagents; D008836:Micrococcal Nuclease; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D003959:Diamines; D005452:Fluoresceins; D007202:Indicators and Reagents; C008697:octamethylenediamine; D001710:Biotin; D004247:DNA; D000469:Alkaline Phosphatase; D008836:Micrococcal Nuclease; D010727:Phosphoric Diester Hydrolases; D019793:Fluorescein