DNA Polymerase Recognition of Unnatural Base Pair: 1-Methoxy-3-methylbenzene and 6-Methyl-1(2H)-isoquinolinone

Abstract

The faithful replication of DNA by DNA polymerases is necessary for the expansion of both the genetic alphabet and the genetic code. 1 The stability and sequence specificity of a natural DNA duplex are based on the complementary and orthogonal Watson-Crick hydrogen-bonding patterns of dA:dT and dC:dG base pairs. Although inter-nucleobase hydrogen bonding and shape complementarity are important for the stability and faithful copying of DNA, there is no reason to assume that the pairing of two unnatural bases could not be driven by other intermolecular interactions, for example, those based on hydrophobic interactions and van der Waals forces. Some groups have used this concept to explore the development of unnatural nucleotides bearing nucleobase analogs that pair via hydrophobic interactions and packing forces. 2-5 Recently, Romesberg’s group found that the heteropair formed between 1-methoxy-3-methylbenzene 1 and 6-methyl-1(2H)-isoquinolinethione 2 is a very promising, predominantly hydrophobic base pair. This base pair is synthesized by the insertion of one unnatural triphosphate opposite the other unnatural nucleotide in the template, and it is then extended by the insertion of the next correct dNTP (natural triphosphates), with relatively high efficiency and fidelity, by diverse DNA polymerases (Figure 1). 5 However, the slowest step in the replication of DNA containing this heteropair is the incorporation of 1 triphosphate opposite 2 in the template. Also, dG inserts competitively opposite 2 in the template, which may limit the use of this heteropair in biological and biotechnological applications. We are therefore interested in the modifications of 1 or 2 to optimize the formation of unnatural heteropairs. Herein, we report the DNA replication results for two nucleotides, 6-methyl-1(2H)-isoquinolinone 3, in which an oxygen atom is substituted for the sulfur atom in 2 to compare the effect of the oxygen atom on replication properties, and 7-methyl-1(2H)isoquinolinone 4, in which, as well as replacing the sulfur atom in 2 by an oxygen atom, the methyl substituent is moved from position 6 to position 7. The substitution at position 7 is expected to bury the methyl group within the duplex, as shown