Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

Abstract

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O-P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH₂-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3'-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA─NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Figure 1.

Structure-guided redesign of 9°N DNA polymerase to catalyze the formation of internucleotidyl N-P bonds. (A) Model of 9°N bound to a primer/template complex. (B,C) Insets show the positioning of the primer and the incoming nucleoside triphosphate within (B) the WT 9°N (PDB accession codes: 5OMV and 5OMQ) and (C) its quadruple-mutant variant 9°N-NRQS (Rosetta based model). Sites of substitutions in 9°N-NRQS are highlighted by red spheres. Active site metal ions are shown: (B) Mg²⁺ in green and (C) Mn²⁺ in purple. Residues are drawn as sticks with color by atom (carbon, white; nitrogen, blue; oxygen, red; phosphorus, orange). (D,E) Template-directed primer-extension reactions catalyzed by (D) WT 9°N and (E) 9°N-NRQS. Chemical structure of (D) natural phosphodiester-linked DNA and (E) N3′ → P5′ phosphoramidate-linked DNA (NP-DNA).

Figure 2.

Analysis of 3′-nucleophile positioning using molecular dynamics (MD) simulations. (A) 2D scatter plots of the in-line attack angle (τ, the angle between the 3′-nucleophile, scissile phosphorus atom, and O5′) and the distance between the 3′-nucleophile and scissile phosphorus atom (r), obtained from MD simulations of 9°N in various active site configurations. In total, eight different active site scenarios are explored: the four panels correspond to simulations in which residue 404 is Asp and the A-site metal ion is present (top left) or absent (top right), residue 404 is Asn and the A-site metal ion is present (bottom left) or absent (bottom right). In each panel, the red and blue colors correspond to the primer being 3′-OH (red) or 3′-NH₂ (blue). The data points are classified as active (A) or non-active (NA), where active configurations are defined as those in which the active site possesses in-line fitness (τ > 150°) and in which the 3′-nucleophile is favorably positioned for catalysis (distance between 3′-nucleophile and P ≤ 3.5 Å). The inset bars represent percentage of favorable configurations of the 3′-nucleophile for catalysis via in-line attack. (B) MD snapshot of the 9°N-N active site showing a representative configuration with high in-line fitness (τ ~ 173°, solid black line). The distance between the 3′-amino nucleophile and D542 is 3.0 Å, suggesting that D542 may serve as a base. Observed hydrogen bond and charge interactions in the active site are highlighted (residues in sticks).

Figure 3.

Rosetta modeling-guided double and combinatorial mutagenesis of 9°N-N. (A) Classification of variants based on the site of amino acid substitution and combinatorial status. Substitutions leading to an increase in NP-DNA polymerase activity are highlighted in green, the variant with the highest catalytic rate is highlighted in blue. (B–G) Rosetta-based structural models of selected key substitutions, highlighting the second-shell effects on the catalytic site (B,C) and the distal finger subdomain (D–G). Active site metal ion Mn²⁺ is in purple. Residues are drawn as sticks with color by atom (carbon, white; nitrogen, blue; oxygen, red; phosphorus, orange). Dashed yellow line indicates the second shell interaction.

Figure 4.

Enzymatic DNA and NP-DNA primer-extensions. (A) Primer-extension reaction conditions: 1 μM 3′-amino-G primer, 1 μM DNA template, 100 μM 3′-NH₂-ddNTP, 1 mM M²⁺ (Mg²⁺ or Mn²⁺), 1 μM polymerase (9°N, 9°N-N, or 9°N-NRQS) in 1× ThermoPol buffer at pH 8.8 at 55 °C. 5′-end of the primer strand is labeled with 6-carboxyfluorescein (FAM), represented as the green sphere. (B) Summary of primer-extension reaction rate, k_obs (h⁻¹). For entries 3 and 4, an upper bound of the k_obs is provided. a: N.D.: not determined. Product formation is complete in less than 1 min. For 9°N-catalyzed dGTP addition in the presence of Mg²⁺, k_pol was measured as 183,600 h⁻¹ (see ref 35); b: reaction temperature is 65 °C. For entries 5–13, R² values for the fits are 0.942, 0.991, 0.967, 0.991, 0.913, 0.997, 0.992, 0.994, and 0.977. (C) Substrate concentration-dependent k_obs vs 3′-NH₂-ddGTP concentration for 9°N-N (red) and 9°N-NRQS (dark gray) at 55 °C. Comparison of maximum rates of 3′-amino-G incorporation, k_pol, and dissociation constants, K_d, for both 9°N-N and 9°N-NRQS.

Figure 5.

9°N-NRQS-catalyzed extensions of the 3′-amino-terminated primer on homopolymeric DNA templates. (A) Primer-extension reaction condition. Four different reaction mixtures were prepared using a specific 3′-NH₂-ddNTP (N: base incorporated to the primer). Y: template base; q: overhang. (B) Denaturing SDS-PAGE analyses of the extension reaction mixtures containing 1 μM 3′-amino-G primer, 1 μM DNA template, 100 μM 3′-NH₂-ddNTP, 1 mM Mn²⁺, and 1 μM 9°N-NRQS in 1× ThermoPol buffer at pH 8.8; for each reaction, samples were quenched at the designated time points. Imaging performed at FAM excitation (epi-blue, 460–490 nm). (C) Plot of the percentage of the unreacted primer strand versus incubation time. The observed rate constants, k_obs (h⁻¹), was calculated using a single exponential decay fit. Error estimates were calculated from the standard mean error of three replicates. R² values for the fits are 0.998 (3′-NH₂-ddTTP), 0.995 (3′-NH₂-ddATP), 0.993 (3′-NH₂-ddCTP), and 0.977 (3′-NH₂-ddGTP).

Figure 6.

Effect of template on 9°N-NRQS-catalyzed extensions of the 3′-amino-terminated primer. (A) Investigation of the NP-DNA polymerase activity using the nucleotide-template mismatch assays at 55 °C (top) and 65 °C (bottom) after 24 h of incubation. Each reaction contained 1 μM 3′-amino-G primer, 1 μM DNA template, 100 μM 3′-NH₂-ddNTP, 1 mM Mn²⁺, and 1 μM 9°N-NRQS in 1× ThermoPol buffer at pH 8.8. Selectivity ratio (SR) for each template-nucleotide combination was calculated based on the ratio of [total band intensity of the extension products for a given 3′-NH₂-ddNTP]/[total band intensity of all extension products combined]. Heatmaps range from red (minimum) to blue (maximum), with values normalized to the lowest SR as 1.0 in each row separately. (B) Investigation of the polymerase activity of 9°N-NRQS on templates with mixed sequences. Reaction mixtures (i–iv) contained 1 μM 3′-amino-G primer, 1 μM DNA template, 200 μM each of 3′-NH₂-ddNTP or dNTP, 1 mM Mn²⁺, and 5 μM 9°N-NRQS in 1× ThermoPol buffer at pH 8.8. Reaction temperature: 65 °C.