Enzymatic Incorporation in DNA of 1,5-Anhydrohexitol Nucleotides

Abstract

The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.

Direct observation of two cyclohexenyl (CeNA) ring conformations in duplex DNA

“…change of the nucleic acids during the elongation process, a few nucleotides away from the active site. [8][9][10] CeNA might therefore be an excellent candidate to generate a polymerase that may copy DNA into CeNA and vice versa. Further investigations will demonstrate if it will be easier to evolve a polymerase for the synthesis of CeNA than for other six-membered modified nucleic acids.…”

Enzymatic Incorporation of LNA Nucleotides into DNA Strands

“…DNA/RNA polymerases incorporate their natural substrates with high specificity and fidelity; however, different research groups have shown that polymerases can also accept a range of sugarmodified nucleotides as substrates. [10][11][12][13][14][15][16][17][18][19][20][21] Although LNA monomers structurally mimic RNA, they mediate high-affinity recognition of complementary RNA and DNA as constituents of either RNA or DNA strands. We therefore decided to study the enzymatic incorporation of LNA monomers into both RNA (transcription experiments) and DNA (primer extension assays) strands by using a variety of DNA/RNA polymerases.…”

Synthesis and Conformational Analysis of 1-[2,4-Dideoxy-4-C-hydroxymethyl-α-l-lyxopyranosyl]thymine

“…6,7 Antiviral activity 1,2,3 has been found in the hexitol series (1) (Figure 1). These molecules are characterized by the presence of an axial base moiety in the most stable conformation.…”