Nucleic Acids Research, 2002, Vol. 30, No. 9 1869-1878
© 2002 Oxford University Press
Synthesis and recognition by DNA polymerases of a reactive nucleoside, 1-(2-deoxy-ß-D-erythro-pentofuranosyl)-imidazole-4-hydrazide
Institut Pasteur, Unité de Chimie Organique URA CNRS 2128, 28 rue du docteur Roux, 75724 Paris cedex 15, France and 1Evologic SA, 89 rue Henri Rochefort, 91000 Evry, France
We report the synthesis of a new nucleoside, 1-(2-deoxy-ß-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dYNH2) as a reactive monomer for DNA diversification. The 5'-triphosphate derivative (dYNH2TP, 1) was evaluated in vitro as a substrate for several DNA polymerases. Primer extension reactions showed that dYNH2TP was well tolerated by KF (exo–) and Vent (exo–) DNA polymerases. One dYNH2MP was incorporated opposite each canonical base with an efficiency depending on the template base (A 
T > G > C). Significant elongation after YNH2 incorporation was observed independently of the YNH2:N base pair formed. When the nucleobase YNH2 was incorporated into synthetic oligodeoxynucleotides via the phosphoramidite derivative 11, it directed the insertion of natural bases as well as itself. The mutagenicity of dYNH2TP was evaluated by PCR amplification using Vent (exo–) DNA polymerase. The triphosphate dYNH2TP was preferentially incorporated as a dATP or dGTP analogue and led to misincorporations at frequencies of 
2 x 10–2 per base per amplification. A high proportion of transversions with a large distribution of all possible mutations was obtained. The reactivity of the nucleobase YNH2 within a template with several aldehydes was demonstrated.
* To whom correspondence should be addressed. Tel: +33 140 61 33 28; Fax: +33 145 68 84 04; Email: spochet{at}pasteur.fr