Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences

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

Anatomical localization of significant differences in DTI and TDI measurements between UCPPS patients (<i>N = 45</i>) and HCs (<i>N = 56</i>).

<p>A) Observed differences in mean diffusivity (MD). B) Observed differences in fractional anisotropy (FA). C) Observed differences in fiber track density. D) Observed differences in generalized anisotropy (GA). Significant clusters were determined by thresholding based on level of statistical significance (<i>P < 0</i>.<i>05</i>) and cluster-based corrections using random permutation analysis. Left column illustrates differences projected onto representative white matter fiber tracts.</p

Anatomical regions and corresponding cluster volumes showing significant differences in MD between UCPPS patients and HCs (HCs).

<p>Minimum cluster size = 171 uL from permutation analysis.</p

Anatomical regions and corresponding cluster volumes showing significant differences in fiber track density on TDI between UCPPS patients and HCs (HCs).

<p>Minimum cluster size = 111 uL from permutation analysis.</p

Correlation matrix between mean MD measurements and MAPP symptom scores in UCPPS patients, localized to ROIs identified as different between UCPPS and HCs on statistical parameter maps.

<p>Dendrograms on the left side of the correlation matrix show hierarchical clustering of ROIs based on the association between MD measurements and symptom scores. Images showing ROI localization are chosen for select associations. Up arrows within cells denote significantly positive correlations (<i>P<0</i>.<i>05</i>) and down arrows within cells denote significantly negative correlations (<i>P<0</i>.<i>05</i>). (Note the level of significance was not corrected for multiple comparisons in this exploratory analysis).</p

Sex differences in fiber track density within A) UCPPS patients, B) positive control patients with IBS, and C) healthy control (HC) participants.

<p>Significant clusters were determined by thresholding based on level of statistical significance (<i>P < 0</i>.<i>05</i>) and cluster-based corrections using random permutation analysis.</p

Correlation matrix between mean track density measurements and MAPP symptom scores in UCPPS patients, localized to ROIs identified as different between UCPPS and HCs on statistical parameter maps.

<p>Dendrograms on the left side of the correlation matrix show hierarchical clustering of ROIs based on the association between track density measurements and symptom scores. Images showing ROI localization are chosen for select associations. Up arrows within cells denote significantly positive correlations (<i>P<0</i>.<i>05</i>) and down arrows within cells denote significantly negative correlations (<i>P<0</i>.<i>05</i>). (Note the level of significance was not corrected for multiple comparisons in this exploratory analysis).</p

Anatomical localization of significant differences in DTI and TDI measurements between UCPPS patients (<i>N = 45</i>) and positive control patients with IBS (<i>N = 39</i>).

<p>A) Observed differences in mean diffusivity (MD). B) Observed differences in fractional anisotropy (FA). C) Observed differences in fiber track density. D) Observed differences in generalized anisotropy (GA). Significant clusters were determined by thresholding based on level of statistical significance (<i>P < 0</i>.<i>05</i>) and cluster-based corrections using random permutation analysis. Left column illustrates differences projected onto representative white matter fiber tracts.</p

Sex differences in mean diffusivity (MD) within A) UCPPS patients, B) positive control patients with IBS, and C) healthy control (HC) participants.

<p>Significant clusters were determined by thresholding based on level of statistical significance (<i>P < 0</i>.<i>05</i>) and cluster-based corrections using random permutation analysis.</p

Sex differences in fractional anisotropy (FA) within A) UCPPS patients, B) positive control patients with IBS, and C) healthy control (HC) participants.

<p>Significant clusters were determined by thresholding based on level of statistical significance (<i>P < 0</i>.<i>05</i>) and cluster-based corrections using random permutation analysis.</p