The strength of the template effect attracting nucleotides to naked DNA

Abstract

The transmission of genetic information relies on Watson-Crick base pairing between nucleoside phosphates and template bases in template-primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template-primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd's range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.

Figure 1.

Binding equilibrium between nucleotide and primer–template duplex; B, B′ = nucleobases.

Figure 2.

Nucleotides and hairpins used for NMR titration. Loops are hexaethylene glycol linkers (HEG).

Figure 3.

Typical results from NMR titrations. (a) Overlay of spectra with ¹H-NMR signal of H-8 proton of the 5′-terminal A residue of hairpin 5′-ATGC(HEG)GCA (2a) (0.5 mM) with increasing concentration of TMP (1t) (0 to 774 mM) at 20°C. (b) Plot of chemical shift displacement upon addition of TMP (1t). See Supplementary Figures S8, S9 and S12–S14 (Supplementary material) for additional spectra and plots of chemical shifts.

Figure 4.

Binding equilibria underlying the inhibitory effect of a free nucleotide on chemical primer extension.

Figure 5.

Oligonucleotide sequences and nucleotides for template-directed primer extension reaction in the presence or absence of an unactivated (free) deoxynucleotide as inhibitor. Assays at increasing concentrations of inhibitor were performed in the presence or the absence of a downstream-binding oligonucleotide that provides additional stacking interactions to the incoming nucleotide. Conditions: 3.6 or 7.2 mM monomer, 0–72 mM free nucleotide, primer extension buffer (200 mM HEPES, 80 mM MgCl₂, 400 mM NaCl, pH 8.9), 20°C.

Figure 6.

Binding of deoxynucleotides to primer–template complexes reveals itself through inhibition of primer extension. Kinetics of extension of primer 5′-CGCACGA-NH₂-3′ (8a) by OAt-esters of deoxynucleotides as templated by TNT-type sequences, where the templating base N is A, C, G or T at increasing concentrations of unactivated dNMP (1a–t) as inhibitor, in the absence of a downstream-binding oligonucleotide at 20°C. Conditions: 36 μM primer, 3.6 or 7.2 mM dNMP-OAt (7a–t), 200 mM HEPES buffer, pH 8.9, 400 mM NaCl, 80 mM MgCl₂. Symbols are experimental data and lines are monoexponential fits.

Figure 7.

Association constants for dNMPs binding to primer–template duplexes displaying complementary base at 20°C, as obtained by averaging over values of NMR titrations and inhibitory studies (first nine entries of Table 1 and entries 1–20 of Table 2). See Tables 1 and 2 for conditions.

Figure 8.

Occupancy of extension site by the deoxynucleotide complementary to the templating base at 20°C, as calculated for different concentrations of 2′-deoxynucleotides 1a–t using binding constants reported in Table 1 or Table 2. Binding to (a) hairpins 2a, 2c, 2g or 4t, (b) template–primer duplexes 8tnt:9a–t and (c) template–primer duplexes 8tnt:9a–t in the presence of downstream-binding oligonucleotide 10a–t. Note the different scales of the x-axes in (a) and (b)/(c).

Figure 9.

Correlation between dissociation constants and rates of extension (determined for 3.6 mM concentration of dAMP-OAt 7a, dCMP-OAt 7c and dGMP-OAt 7g, or 7.2 mM concentration of TMP-OAt 7t in the presence of downstream-binding oligonucleotides 10a–t at 20°C) for the templating sequences listed in Table 2. The lines are linear functions obtained using regression analysis; solid black line, all 16 values (r² = 0.775), broken line: highest data point excluded (r² = 0.209).

Figure 10.

The role of inhibition in different chemical primer extension scenarios. On the right of each part, kinetics of primer extension (black dots) and monomer hydrolysis (open circles) are shown, and on the left, the calculated occupancy of the extension site by monomer or hydrolyzed monomer (inhibitor) at 0 min is shown as a bar graph. (a) Amino-terminal primer 9a (36 μM), template 8tct, helper strand 10a and 3.6 mM dGMP-OAt (7g); (b) 3′-amino-terminal hairpin 2a (36 μM) and 3.6 mM dTMP-OAt (7t); (c) RNA primer 13g (100 μM), template 12ccg, helper strand 14c and 20 mM rGMP-OAt (15g). The K_d values used for calculating occupancies are 2 mM for dGMP, 260 mM for dTMP and 14 mM for GMP (Tables 1 and 2). See also the Supplementary Material (Supplementary Figures S15b, S22 and S33). Kinetics and hydrolysis data for the RNA case are from reference (29).

Figure 11.

Binding equilibria and reactions for extension of a primer (P₁) by an activated nucleotide as monomer (M). Hydrolysis of the monomer produces a free nucleotide that acts as an inhibitor (Inh). The non-covalent binding of both activated and free nucleotide to the primer are governed by the dissociation constant (K_d), while the rates of extension (k_cov) and hydrolysis (k_h) govern the fate of the monomer. It is assumed that the primer is stably bound to the template.

Figure 12.

Oligonucleotide sequences and ribonucleotides for template-directed RNA primer extension reaction in the presence or absence of an unactivated (free) ribonucleotide as inhibitor, as described (29).

Figure 13.

Simulated time-dependent yield of extended RNA primer (Figure 12) with and without hydrolysis and inhibition at initial concentrations of 20 mM monomer (GMP-OAt) and 268 μM primer. (a) Symbols represent published experimental values (29) for assays with different concentrations of inhibitor (GMP) added; circles: none, triangles 5 mM, diamonds 20 mM GMP. Solid lines are calculated using Equation (6). Values used for the simulation: K_d = 14 mM; k_cov = 0.27 h⁻¹, k_h = 0.147 h⁻¹. The dashed black line shows hypothetical kinetics without formation of inhibitor through hydrolysis. (b) Same as (a) but with a K_Inh value increased by a factor of 3, as expected for this system containing a downstream-binding oligonucleotide.

Figure 14.

Simulated time-dependent yield of extension of amino-terminal DNA primer 9g at decreasing concentration of monomer in the presence of downstream-binding oligonucleotide 10g. Lines are calculated using Equation (6) with k_cov values obtained from rate constants (assuming monoexpoential kinetics) and occupation numbers based on dissociation constants for nucleotides. Filled circles are experimental data for monomer concentrations of 3.6 mM (red), 0.36 mM (blue), 0.18 mM (green) or 0.036 mM (pink). (a) Monomer is A (dAMP-OAt) and template is 8ctc; values of the simulation: K_d = 6.9 mM; k_h = 0.109 h⁻¹, k_cov = 8.3 h⁻¹. (b) Monomer is G (dGMP-OAt) and template is 8ccc; with values of K_d = 6.8 mM; k_h = 0.093 h⁻¹ and k_cov = 12.2 h⁻¹. (c) Same as (b), except that a 2-fold lower monomer concentration was assumed for the pink data at the end of the dilution series (0.018 mM).

Figure 15.

Effect of nucleobase and sequence context on binding of nucleotides to templating bases: heat map representation of representative dissociation constants for complexes between deoxynucleotides and termini of hairpins, primer-template complexes or primer–template complexes with downstream-binding oligonucleotide at 20°C. The color bar on the right-hand side is a graphical definition of how color intensity codes for binding strength. Data are from Tables 1 and 2.