title: N(4)C-alkyl-N(4)C cross-linked DNA: bending deformations in duplexes that contain a -CNG- interstrand cross-link.
abstract: Short DNA duplexes containing a 1,3-N(4)C-alkyl-N(4)C interstrand cross-link that joins the two C residues of a -CNG- sequence were prepared using either a phosphoramidite or convertible nucleoside approach. The alkyl cross-link consists of 2, 4, or 7 methylene groups. The duplexes, which contain a seven-base-pair core and A(3)/T(3) complementary 3'-overhanging ends, were characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Ultraviolet thermal denaturation studies showed that the duplexes denature in a cooperative manner and that the length of the cross-link affects the thermal stability. Thus, the transition temperature of the ethyl cross-linked duplex, 42 degrees C, is 16 degrees C higher than the melting temperature of the corresponding non-cross-linked control, whereas the transition temperatures of the butyl and heptyl cross-linked duplexes, 73 and 72 degrees C, respectively, are 46-47 degrees C higher. The reduced molecularity of denaturation of the cross-linked duplexes versus melting of the non-cross-linked duplex most likely accounts for these differences. Examination of molecular models suggests that the ethyl cross-link is too short to span the distance between the two C residues at the site of the cross-link in B-form DNA without causing distortion of the helix, whereas less and no distortion would be expected for the butyl and heptyl cross-links, respectively. The circular dichroism spectra, which show greatest deviation in the ethyl cross-linked duplex from B-form DNA, are consistent with this expectation. Anomalous mobilities on native polyacrylamide gels of multimers produced by self-ligation of each of the cross-linked duplexes suggest that the ethyl and butyl cross-linked duplexes undergo bending deformations, whereas multimers derived from the heptyl cross-linked duplex migrated normally. The bending angle was estimated to be 20 degrees, 13 degrees, and 0 degrees for the ethyl, butyl, and heptyl cross-linked duplexes, respectively. Thus, it appears that the degree of bending in these N(4)C-alkyl-N(4)C cross-linked duplexes is controlled by the length of the cross-link.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Noronha
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wilds
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
mesh_terms: D000478:Alkylation; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D006358:Hot Temperature; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D013816:Thermodynamics
chemical_list: D003432:Cross-Linking Reagents; D009692:Nucleic Acid Heteroduplexes; D004247:DNA