Tuning the reaction site for enzyme-free primer-extension reactions through small molecule substituents.
The replication of genetic information relies on the template-directed extension of DNA primers catalyzed by polymerases. The active sites of polymerases accept four different substrates and ensure fidelity and processivity for each of them. Because of the pivotal role of catalyzed primer extension for life, it is important to better understand this reaction on a molecular level. Here we present results from primer-extension reactions performed with chemical systems that show high reactivity in the absence of polymerases. Small molecular caps linked to the 5'-terminus of templates are shown to enhance the rate and selectivity of primer extension driven by 2-methylimidazolides as activated monomers for any of the four different templating bases (A, C, G, and T). The most consistent effect is provided by a stilbene carboxamide residue, rather than larger aromatic or aliphatic substituents. Up to 20-fold rate enhancements were achieved for the reactions at the terminus of the template. The preference for a medium size cap can be explained by competing interactions with both the oligonucleotides and the incoming deoxynucleotide. The data also show that there is no particularly intractable problem in combining promiscuity with fidelity. Exploratory experiments involving a longer template and a downstream-binding strand with a 5'-cap show up to 38-fold rate acceleration over the same reaction templated by a single overhanging nucleotide.
Rojas Stütz
Jan A
JA
Institut für Organische Chemie, Universität Karlsruhe TH, 76131 Karlsruhe, Germany.
Richert
Clemens
C
D024506:5' Flanking Region; D017931:DNA Primers / Q000737:chemistry*; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D013267:Stilbenes / Q000737:chemistry; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D017931:DNA Primers; D007093:Imidazoles; D013267:Stilbenes; D004259:DNA-Directed DNA Polymerase