title: Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine.
abstract: The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for T(m) and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct T(m)'s when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T(m) closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Pascal A
authors.initials: PA
authors.lastname: Küpfer
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Leumann
mesh_terms: D020029:Base Pairing; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*
chemical_list: D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; C434331:phosphoramidite; C017400:5-hydroxycytosine; D003596:Cytosine; D012194:RNA-Directed DNA Polymerase