Differential Discrimination of DNA Polymerases for Variants of the Non-Standard Nucleobase Pair Between Xanthosine and 2,4-Diaminopyrimidine, Two Components of an Expanded Genetic Alphabet

Abstract

Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms. Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c7dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA). Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4-diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c7dX was significantly lower and also independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E. coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c7dX.

Nucleic acid duplexes incorporating a dissociable covalent base pair

“…Experiments in which EDC or MgCl 2 was omitted were used as controls. A standard ligation experiment with unmodified oligonucleotides GL 5 and Im(p*L 3 p) aligned on the unmodified template Tem was carried out under identical conditions to permit comparison between a standard ligation reaction and ligation to a covalent base pair. A ligation reaction between TemAF and p*L 5 (Fig.…”

“…In Fig. 5, lane 9, we show the product p*TemAL 5 p*F from a ligation reaction between 5Ј-32 P-labeled TemAF and activated 5Ј-32 P-labeled L 5 and, in Fig. 5, lane 10, the product from the corresponding reaction with the reduced Fig.…”

“…The sugar-phosphate backbones of RNA and DNA have been replaced by a variety of alternative backbones (1-4), a number of alternative or additional base pairs have been described (5,6), and the possibility of using shape analogues of the bases has been explored (7,8). Cross-linking of nucleic acid strands has also been discussed in many publications (9)(10)(11)(12)(13)(14), but, as far as we are aware, reversible cross-linking leading to a ''covalent base pair'' without distortion of the structure of the double helix has not been described.…”

“…5, lane 3). We carried out a parallel standard ligation reaction using Ϸ0.7 equivalent of p*L 3 p and a 10-fold excess of GL 5 to guarantee that the 5Ј-end of the template Tem was fully occupied. The yield of the standard ligation product was only 7% (Fig.…”

Oligonucleotides Containing 7-Deaza-2′-deoxyxanthosine: Synthesis, Base Protection, and Base-Pair Stability

“…It has been reported that 2'-deoxyxanthosine (1) might expand the genetic code when it pairs with a pyrimidine-2,4-diamine C-nucleoside [14]. However, experimental data show that the enzymatic incorporation of a pyrimidine-2,4-diamine C-nucleoside triphosphate on a DNA template containing compounds 1 or 3 is slow compared to the canonical nucleotides [14].…”