posted on 2013-03-19, 00:00authored byAlessandro Calabretta, Christian J. Leumann
Two
RNA phosphoramidites containing the bases 1,N6-ethenoadenine
(εA) and 3,N4-ethenocytosine (εC) were synthesized.
These building blocks were incorporated into two 12-mer oligoribonucleotides
for evaluation of the base pairing properties of these base lesions
by UV melting curve (Tm) and circular
dichroism measurements. The Tm data of
the resulting duplexes with the etheno modifications opposing all
natural bases showed a substantial destabilization compared to the
corresponding natural duplexes, confirming their inability to form
base pairs. The coding properties of these lesions were further investigated
by introducing them into 31-mer oligonucleotides and assessing their
ability to serve as templates in primer extension reactions with HIV,
AMV, and MMLV reverse transcriptases (RT). Primer extension reactions
showed complete arrest of the incorporation process using MMLV RT
and AMV RT, while HIV RT preferentially incorporates dAMP opposite
εA
and dAMP as well as dTMP opposite εC. The properties of these
RNA lesions are discussed in the context of its putative biological
role.