Cascade of reduced speed and accuracy after errors in enzyme-free copying of nucleic acid sequences

Abstract

Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

Figure 1. Representative primer extension assay by MALDI-TOF mass spectra

Shown are a fast extension (A: 2a with template 1tt and 3a-t) and a slow and inaccurate extension (B: 2t with template 1tc and 3a-t).

Figure 2. Stalling factors for different systems

Shown here are the (A) DNA primer, DNA template system with OAt esters and (B) different template backbone and leaving groups. The stalling factor is the ratio of polymerization rate from matched terminus to rate of extension from mis-matched terminus. Stalling factors are calculated for extension by the correct monomer (S_G); by definition, S_G = 1 for extension after a matched base pair. In (B), white is template 5uu with 4a-t; black is 5uu with 3a-t; hatched is 1tt with 4a-t; gray is 1tt with 3a-t (also shown in (A)).

Figure 3. Representative primer extension assay by PAGE

Shown are a fast extension (A: 7g on template 6.0ca with 8u) and a slow extension from a mismatch (B: 7t on template 6.0uc with 8g); lines are drawn to connect successive data points.

Figure 4. Recovery of extension rate following a single mismatch

Distance (x-axis) is the number of bases from the mismatch to the end of the primer before reaction, including the mismatch itself (‘0’ is no mismatch; ‘1’ is incorporation immediately after the mismatch, etc). Black circles are experimental values, averaged over replicates, for several primer-template complexes, where the relative rate is k/k₀ (k is the observed rate and k₀ is the rate with no mismatch). Black line is the average among complexes for experimental values at the same distance. Gray triangles are theoretical values for different primer-template complexes, where the relative rate is equal to (P_ck_t + P_ok_n)/k_t, where P_c and P_o are the probabilities that the base pair is closed or open, respectively, and k_t and k_n are the rates of templated and non-templated extension, respectively. Gray line is the average among different complexes for theoretical values at the same distance. See Supporting Table S1 for experimental rates and errors.

Figure 5. Mutation frequencies

Data are shown for (A) the RNA system after single mismatches, and (B) the LNA/DNA system (Supporting Table S5). Patterns correspond to the activated monomers as follows: black = C, gray = G, hatched = A, white = U.

Figure 6. Extension rate vs. number of mutations

Average values for different mismatches are shown as different points, with standard deviation indicated by error bars. See Table 3 and Supporting Table S2 for details.

Figure 7. The effect of sequence context on rate (A) and fidelity (B)

Template sequences are from series 6.0 (A) and series 1 (B). The templating base is positioned across from the incoming nucleotide; the adjacent 5′ and 3′ bases are positioned immediately 5′ and 3′of the templating base, respectively. Heat maps are shown with each square shaded according to the relative values given in each square (higher value = darker square), for first-order rate constants (h^-1) in (A) or fidelities f_G in (B). Template is given in parentheses. Activated monomers are indicated at the bottom of panel B. See Supporting Tables S3-4 for more details.

Figure 8. Computed effect of the cascade of mutations on copying of 50-mers

Histograms are shown for (A) number of errors per sequence, (B) average size of error clusters per sequence, and (C) relative polymerization time compared to a perfect copy (T_r).

Scheme 1. Experimental systems for non-enzymatic extension

(A) DNA template and primer; (B) RNA template, DNA primer. All primer sequences contain a 3′-amino-2′,3′- dideoxynucleotide as the 3′ terminal nucleotide.

Scheme 2. Experimental systems for non-enzymatic extension (continued)

(A) RNA template, RNA primer; (B) mixed DNA/LNA template, DNA primer. All primer sequences contain a 3′-amino-2′,3′-dideoxynucleotide as the 3′ terminal nucleotide.

Scheme 3

Templated (A) and non-templated (B) primer extension with activated 2′-deoxynucleoside 5′-monophosphates, with nucleophilic attack of the primer terminus and release of the leaving group. The degree of stalling of extension after a mismatch is limited by the background rate of non-templated extension. R = H or OH; B and B′ are nucleobases.

Scheme 4

Model reaction performed for determining the rates of template-free reaction between an activated deoxynucleoside-5′-monophosphate and an aminoterminal nucleic acid (LG = leaving group, B = nucleobase).

Scheme 5. Reaction scheme for the simulation

Polymerization proceeds along an arbitrary template sequence. Reaction (1) is a possible initiation point for a cascade of errors. If an incorrect nucleotide is incorporated, the fidelity and the speed of the following reactions are reduced. If reaction (2) is followed by a correct incorporation, extension occurs at a reduced reaction rate in reactions (3) and (5) until the normal rate is recovered. More likely, (2) is followed by another error, which leads to an even stronger reduction in the rate of reaction (4) and a slower recovery in reaction (6) and subsequent steps. Yet another error in (4) leads to a slow reaction (7) and so forth.