Base Pairing and Miscoding Properties of 1,N6-Ethenoadenine- and 3,N4-Ethenocytosine-Containing RNA Oligonucleotides

Two RNA phosphoramidites containing the bases 1,N-6-ethenoadenine (epsilon A) and 3,N-4-ethenocytosine (epsilon C) 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 (T-m) and circular dichroism measurements. The T-m 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 epsilon A and dAMP as well as dTMP opposite epsilon C. The properties of these RNA lesions are discussed in the context of its putative biological role.