Templating efficiency of naked DNA

Abstract

Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with >or=93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows >or=72% of the correct extension product in all cases, and >or=90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

Fig. 1.

Copying of DNA involves the stepwise extension of a primer as directed by the sequence of the template. The grahic shows the molecular components for polymerase-catalyzed copying (1 and 2) or enzyme-free copying (3 and 4). B, bases of template; B′, bases of primer/monomer.

Scheme 1.

Sequences of assays with (A) or without (B) downstream-binding strand. Core triplets are given as B¹BB², where B is the templating base.

Fig. 2.

Representative kinetics of primer extension for template core sequences B¹BB² (A) TCT, and (B) AAA. Lines are monoexponential fits (symbols); gray, extended primer; black, primer.

Fig. 3.

“Heat map” representation of the rates of primer extension for different core triplets of the template. The deeper red the color, the faster the reaction. The legend on the right shows the color code used for each box; t_1/2, time for extension of 50% of the primer.

Scheme 2.

Proposed mechanism of primer extension.

Fig. 4.

Influence of concentration of the incoming monomer (here TMP-OAt) on the lag phase: kinetics of primer extension on template GAA at 20 °C. Lines are fits using Eqs. 2 and 3 with one common set of kinetic parameters k₁, k₂, k₃.