Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

Nucleic Acids Res. 2011 Nov;39(21):9422-32. doi: 10.1093/nar/gkr673. Epub 2011 Aug 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • Cytosine / analogs & derivatives*
  • Cytosine / chemistry
  • Nucleic Acid Denaturation
  • Oligoribonucleotides / biosynthesis
  • Oligoribonucleotides / chemistry*
  • Organophosphorus Compounds / chemistry
  • Oxidation-Reduction
  • RNA-Directed DNA Polymerase / metabolism*


  • Oligoribonucleotides
  • Organophosphorus Compounds
  • phosphoramidite
  • 5-hydroxycytosine
  • Cytosine
  • RNA-Directed DNA Polymerase