Synthesis, characterization and solution structure of tethered oligonucleotides containing an internal 3'-phosphoglycolate, 5'-phosphate gapped lesion

Abstract

Bleomycins (BLMs) are antitumor antibiotics that in the presence of iron and oxygen mediate DNA damage by 4'-hydrogen atom abstraction of pyrimidines 3' to guanines. The resulting 4'-deoxyribose radicals can be trapped by O2 and ultimately result in the formation of base-propenal and gapped DNA with 3'-phosphoglycolate (3'-PG) and 5'-phosphate (5'-P) ends. The role of this lesion in triggering double-strand cleavage of duplex DNA by a single BLM molecule and the mechanism by which this lesion is repaired in vivo remain unsolved problems. The structure of these lesions is an essential step in addressing both of these problems. Duplex DNAs (13mers containing tethered hexaethylene glycol linkers) with GTAC and GGCC cleavage sites have been synthesized in which gaps containing 3'-PG and 5'-P ends at the sites of BLM cleavage have been inserted. The former sequence represents a hot spot for double-strand cleavage, while the latter is a hot spot for single-strand cleavage. Analytical methods to characterize the lesioned products have been developed. These oligonucleotides have been examined using 2D NMR methods and molecular modeling. The studies reveal that the lesioned DNAs are B-form and the 3'-PG and 5'-P are extrahelical. The base opposite the gap and the base pairs adjacent to the gap remain well stacked in the DNA duplex. Titrations of the lesioned GGCC oligomer with HOO-CoBLM leads to a mixture of complexes, in contrast to results of a similar titration with the lesioned GTAC oligomer.

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

“…To investigate the influence of neighboring bases on the conformation of the abasic site, we also attempted to apply 2D NMR spectroscopy to obtain structural information of the abasic site in the sequence of 5′-CCAAGG X CAGGG-3′/3′-GGTTCC G GTCCC-5′. The 5′-GG X C-3′/3′-CC G G-5′ sequence was chosen for direct comparison to our previous structural studies of the 3′-phosphoglycolate/5′-phosphate lesion in the same sequence ( 41 ). In contrast with the 5′-G X AC-3′ sequences, we could not assign most protons associated with the neighboring bases (G6 and G17-C19, see Supplementary Table S4 for partial proton assignment).…”

Aerobic Fate of the C-3‘-Thymidinyl Radical in Single-Stranded DNA

“…Reference oligomers 3′-P (4), 5′-P (12), and 5′-P-1 (11) were purchased from Synthegen and Integrated DNA Technologies. 3′-PGA (2) and 3′-PG (3) were synthesized from the corresponding glycerol-terminated oligonucleotide by established methods (20,21). The glycerol-terminated oligomer was synthesized by the Hphosphonate method beginning with 3-[(4′,4-dimethoxytrityloxy)glyceryl-1-succinoyl]-long chain alkylamino controlled pore glass columns from Glen Research.…”

NMR Structures of Damaged DNA

“…The imino proton signals of gap-flanking base pairs are significantly broaden, due to increased solvent exchange, and shift upfield by about 0.5−0.7 ppm. Base pairs next to those flanking the gap are affected also, showing a reduced intensity of their imino proton signals at 25 °C . The different behavior could be attributed to an increase in electrostatic repulsions caused by the charged ends, although variations between duplex sequences 10 and 11 may also play a role.…”