Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (a TNA) for Effective Nonenzymatic Primer Extension

Abstract

Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence of N-cyanoimidazole (CNIm) and Mn²⁺; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd²⁺, Ni²⁺, and Co²⁺ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

Figure 1

(a) Chemical structures of DNA and L-aTNA. (b) Schematic illustration of the chemical ligation reaction by CNIm and a divalent metal cation. (c) Sequences of L-aTNA and DNA used for chemical ligation. (d) Denaturing PAGE analysis of chemical ligation of 8-mer L-aTNA fragments (T8A/T8B-3′p) on the 16-mer template (T16t) in the presence of CNIm and chloride salt of an indicated divalent metal cation (MCl₂). Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 100 mM NaCl, 5 mM MCl₂, 20 mM CNIm, 25 °C. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 4 W at room temperature for 1.5 or 2 h. Negative control (lane 1) included only T8A and NaCl. (e) Yield as a function of time for ligation of T8A to T8B-3′p on the T16t template (left) and of D16A-3′p to D16B on the D32t template (right) in the presence of indicated divalent metal cations. The graphs are based on data shown from panel d and Figure S1. (f) Calculated k_obs values for chemical ligation of indicated oligomers in the presence of indicated metal ions. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 100 mM NaCl, 5 mM MCl₂, 20 mM CNIm, 25 °C. k_obs values were calculated from linearized plots of -ln([Fragment A]/[Fragment A]₀) assuming a pseudo-first-order reaction.

Figure 2

(a) Scheme of reaction of PhP and CNIm in the presence of Cd²⁺. (b) ¹H NMR time course analysis. ¹H NMR peaks colored brown, red, and green were assigned to PhP, CNIm, and Im, respectively. Reaction conditions: 5 mM PhP, CdCl₂, and CNIm, 25 °C. (c) Possible reaction scheme and intermediates (i) suggested by NMR and (ii) not observed. (d) Normalized peak intensity of CNIm (red) and Im (green) calculated from NMR spectra.

Figure 3

(a) Plot of -ln([Fragment A]/[Fragment A]₀) as a function of time over a range of CNIm concentrations. Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 5 mM CdCl₂, 1, 5, 10, or 20 mM CNIm, 25 °C. (b) Apparent k₁ obtained assuming a pseudo-first-order reaction. k_1,app of T8A/T8B-3′p/T16t and the other k_1,apps were calculated from plots shown in Figures 3a and S11–S15, respectively. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 5 mM CdCl₂, 1, 5, 10, or 20 mM CNIm, 25 °C. (c) Plots of apparent k₁ values for each component versus CNIm concentration based on the Ostwald isolation method. (d) Energy-minimized structures of T8B-3′p and template (top) and T8A-1′p and template (bottom) illustrating interactions of phosphate with the neighboring amide group. (e) Correlation between the concentration of Cd²⁺ ions and the rate constant k₁. (f) Schematic illustration of the predicted mechanism involving divalent metal cations in activation of the phosphate group and rates for steps 1 and 2 and the overall reaction.

Figure 4

(a and b) Schematic illustrations of elongation via chemical ligation of an L-aTNA primer in (a) 3′ → 1′ direction and (b) 1′ → 3′ direction. (c) Denaturing PAGE of L-aTNA elongation reaction products in the presence of Cd²⁺ with ligation in the 1′ → 3′ direction. (d) Comparison of yields of full-length elongation products in different conditions. Reaction conditions: 0.9 μM primer, 100 μM T3Bmix, 1.0 μM template, 100 mM NaCl, 5 mM CdCl₂ or 20 mM MnCl₂, 20 mM CNIm, 4 °C for 6 h in the presence of Cd²⁺ or for 24 h in the presence of Mn²⁺. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h, 4 °C, 4 W. Negative control (lane 1) included only primer, NaCl, and divalent metal cation salt (Lane 1). Markers were prepared by the reaction using complementary fragments (Figure S23).

Figure 5

(a) Schematic illustration of chemical primer extension on 23-mer and 29-mer L-aTNA templates with optimized conditions. (b) Denaturing PAGE of the reaction with T23t as the template (left) and T29t as the template (right). Reaction conditions: 0.45 μM Rev-T8A-3′p, 200 μM T3Bmix for T23t or 400 μM T3Bmix for T29t, 1.0 μM T23t or T29t, 100 mM NaCl, 5 mM CdCl₂, 20 mM CNIm, 4 °C for 4 h. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h with T23t as the template or 3.5 h with T29t as the template, 15 °C, 4 W. Negative controls (lanes 1 and 6) included only Rev-T8A-3′p, NaCl, and CdCl₂. (c) MALDI-TOF MS analyses of products of the elongation reaction for 2 h with T23t as the template (left) and for 4 h with T29t as the template (right).