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

6.0cc was particularly fast compared to reactions with the same templating base but different adjacent bases (6.0ac, 6.0gc, 6.0uc).

“6.0cc was particularly fast compared to reactions with the same templating base but different adjacent bases (6.0ac, 6.0gc, 6.0uc).”

In the RNA system, fidelities for copying templating bases G (5%) and U (22%) after a mismatch were similar to or less than random incorporation (25%).

“The fidelities for copying templating bases G (5%) and U (22%) in this context were similar to or even less than that expected from random incorporation (25%).”

Stalling factors Sp, which consider extension by any monomer, are decreased by 2-3-fold compared to SG.

“If one considers extension by any monomer (Sp) rather than only by the correct monomer, stalling factors are decreased by 2-3-fold (Supporting Figure S1A)”

After a single mismatch, copying C had an apparent fidelity of 88%.

“After a single mismatch, copying C had an apparent fidelity of 88% (Figure 5A; Table 3).”

Polymerization times were substantially increased in the simulation.

“Polymerization times were substantially increased (Figure 8C).”

After two consecutive mismatches, the apparent fidelity stayed at a similar level of 89%.

“After two consecutive mismatches, the apparent fidelity stayed at a similar level of 89%.”

Post-mismatch fidelities were similar on DNA template 1tt for OAt esters 3a-t (29%) and imidazolides 4a-t (30%).

“The fidelities were similar on DNA template 1tt (29% for 3a-t, 30% for 4a-t).”

Mis-incorporation rates were generally quite high after a mismatch.

“Mis-incorporation rates were generally quite high after a mismatch.”

At distances of three bases or more, extension proceeded at about the normal rate.

“Extension at distance 3 and greater proceeded at approximately the normal rate.”

The presence of two consecutive mutations decreased the extension rate by an additional factor of ~6 compared to a single mutation.

“The presence of two consecutive mutations decreased the extension rate by an additional factor of ~6 compared to a single mutation (Supporting Table S2, Figure 6).”

For imidazolides, the non-templated reaction is >1100-fold slower than the templated reactions.

“For the imidazolides, the non-templated reaction is >1100-fold slower than the templated reactions.”

Pyrimidine-pyrimidine mismatches produced the largest stalling factors.

“The highest stalling factors were associated with pyrimidine-pyrimidine mismatches.”

Reactions with the highest fidelities contained template 'CC' (1ca, 1ct).

“As seen for absolute rates, the reactions with the highest fidelities contained template ‘CC’ (1ca, 1ct), again suggesting cooperativity among monomers enhancing the reaction efficiency, at least for G monomers (Figure 7B, Supporting Table S4).”

On average, overall post-mismatch fidelity was similar for DNA template 1tt (29%) and RNA template 5uu (25%).

“On average, the overall fidelity was similar for 1tt (29%) and 5uu (25%), although 1tt was more variable (8-47% for different mismatches) than 5uu (20-32%).”

The reaction rate for correct extension in the RNA system was similar to analogous RNA template-DNA primer and DNA template-DNA primer reactions with the same base pair terminus in a different sequence context.

“The reaction rate for correct extension in the RNA system was similar to that for the analogous RNA template - DNA primer (5uu, 2a, 4a-t) and DNA template – DNA primer (1tt, 2a, 4a-t) reactions having the same base pair terminus in a different sequence context (Table 2).”

In the RNA system, overall fidelity after a C/T mismatch was 38%.

“Overall fidelity after a mismatch was just 38%.”

DNA/LNA templates produced a mutation profile most similar to the system with an RNA template and DNA primer.

“The DNA/LNA templates produced a mutation profile that was most similar to the system with an RNA template and DNA primer (Supporting Figure S23).”

The largest extension rates occurred when the template consisted of two identical nucleotides (CC, GG).

“The largest extension rates occurred when the template consisted of two identical nucleotides (CC, GG).”

All modifications did not change the qualitative finding that non-enzymatic template-directed polymerization leads to a segregation of mutations onto full-length sequence copies, such that a small fraction of copies contains a large fraction of the copying errors.

“All of these modifications did not change the qualitative finding that non-enzymatic template-directed polymerization leads to a segregation of mutations onto full-length sequence copies, such that a small fraction of copies contains a large fraction of the copying errors.”

Error clusters consisted of 5 consecutive errors, on average.

“Error clusters consisted of 5 consecutive errors, on average (Figure 8B).”

The greatest stalling occurred immediately after the mismatch.

“The greatest stalling occurred immediately after the mismatch, as described earlier.”

For all systems studied, an initial mismatch was likely to be followed by another error 54-75% of the time.

“Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time)”

Post-mismatch fidelity for DNA template 1tt was more variable (8-47%) than for RNA template 5uu (20-32%).

“On average, the overall fidelity was similar for 1tt (29%) and 5uu (25%), although 1tt was more variable (8-47% for different mismatches) than 5uu (20-32%).”

The relative proportion of incorporation of each of the four possible nucleotides tended to be similar regardless of the identity of the templating base.

“The relative proportion of incorporation of each of the four possible nucleotides tended to be quite similar regardless of the identity of the templating base (Figure 5A).”

When a matched base pair followed the mismatch, the extension rate returned to almost normal.

“If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels.”

Some post-mismatch fidelities in the DNA system were below the expectation for random incorporation (25%).

“Some fidelities were below the expectation for random incorporation (25%).”

The probability that a primer-template complex forms a closed base pair at the terminus, estimated from RNA folding calculations, qualitatively agrees with the relative extension rates measured in the experiments.

“The qualitative behavior of the recovery of extension rate inferred from this probability was in reasonable agreement with the relative extension rates measured in our experiments (Figure 4).”

On RNA template 5uu, post-mismatch fidelity was higher for imidazolides 4a-t (46%) than for OAt esters 3a-t (25%).

“A greater difference was seen on the RNA template 5uu (25% for 3a-t, 46% for 4a-t), largely due to the relatively high fidelity (79%) with primer 2g (a G/U mismatch) using 4a-t, which contrasts with the low fidelity after the same mismatch using 3a-t.”

For the RNA template, imidazolides gave faster reactions than OAt esters, by at most one order of magnitude.

“For the RNA template, the imidazolides gave faster reactions than the OAt esters (by at most one order of magnitude).”

On the DNA template, the identity of the 5′ activated leaving group did not greatly affect absolute rates, within 5-fold.

“On the DNA template, the identity of the 5′ activated leaving group did not greatly affect absolute rates (within 5-fold; Table 2)”

If the correct base occurred after the mismatch, extension proceeded at the next base with a slightly reduced but nearly normal rate, within a factor of 2.

“If the correct base occurred after the mismatch, then extension proceeded at the next base with a slightly reduced, but nearly normal rate (within a factor of 2).”

For OAt ester monomers, the non-templated model reaction is >60-fold slower than the templated reaction.

“For the OAt ester monomers, the non-templated model reaction is >60-fold slower than the templated reaction.”

In the DNA system with OAt esters, observed post-mismatch fidelities ranged from 8-63%, with an overall average fidelity of 31%.

“The observed post-mismatch fidelities ranged from 8-63%, with an overall average fidelity of 31% (averaging over all four possible template bases for a given primer).”

The DNA template gave somewhat faster extension by the correct monomer after a correctly matched terminus compared to the RNA template, by about 4-fold.

“In terms of absolute reaction rates, the DNA template gave somewhat faster extension by the correct monomer after a correctly matched terminus compared to the RNA template (~4-fold; Table 2).”

The presence of an error causes further extension to stall by 1-2 orders of magnitude for a DNA primer (on both a DNA or RNA template) and for an RNA system.

“We found that the presence of an error causes further extension to stall by 1-2 orders of magnitude for a DNA primer (on both a DNA or RNA template) and for an RNA system, supporting prior work.23”

• cites [23] Rajamani S., 2010, J. Am. Chem. Soc. sourceNot checked yet

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The non-templated model reaction of activated monomers proceeds at a slow but measurable rate.

“The reaction proceeds at a slow but measurable rate (Table 5).”

Decreasing the importance of stalling by setting the non-templated reaction rate to be at least 10% of the incorporation rates decreased the mean polymerization time by roughly 2-fold (average Tr = 9).

“Decreasing the importance of stalling by setting the non-templated reaction rate to be at least 10% of the incorporation rates decreased the mean polymerization time by roughly 2-fold (average Tr = 9).”

The extension rate was essentially normal shortly after a single mismatch.

“The extension rate was essentially normal shortly after the mismatch.”

Extension from mismatches in the RNA system was 1-2 orders of magnitude slower than from the matched terminus.

“Extension from the mismatches (6.1a-u) was between 1-2 orders of magnitude slower than using 6.0uc.”

After one mismatch, extension was typically 10 to 100 times slower than error-free extension.

“and extension after a single mismatch was generally 10-100 times slower than extension without errors.”

Stalling factors did not appear to be affected systematically by the identity of the leaving group.

“Stalling factors did not appear to be affected systematically by the identity of leaving group (Figure 2B; Supporting Figure S1B).”

The identity of the leaving group did not appear to have a systematic effect on the fidelities.

“The identity of the leaving group was also altered for two reactions using 3a-t instead of 4a-t, but this did not appear to have a systematic effect on the fidelities.”

In the model background reaction, imidazolides reacted more slowly than OAt esters.

“In the model background reaction, imidazolides reacted more slowly than OAt esters, suggesting that greater accuracy could be eventually obtained in a system using imidazolides.”

Downstream extension rates correlated well with the predicted base-pairing probability for the terminal base pair.

“Indeed, the downstream extension rates correlated well with the predicted base-pairing probability for the terminal base pair.”

Incorporation after a mismatch was extremely error-prone in all systems tested, with fidelity close to that of random incorporation (25%).

“Incorporation after a mismatch was extremely error-prone in all systems tested, with fidelity (i.e., frequency of correct incorporation) close to that of random incorporation (25%).”

The presence of an isolated single mismatch did not affect extension rates farther downstream, if the mismatch were followed by a correct Watson-Crick base pair.

“The presence of an isolated single mismatch did not affect extension rates farther downstream, if the mismatch were followed by a correct Watson-Crick base pair.”

Reaction rates varied over two orders of magnitude for different sequence contexts in the template.

“Reaction rates varied over two orders of magnitude for different sequence contexts in the template.”

High post-mismatch fidelity tended to occur across templating base C.

“High post-mismatch fidelity tended to occur across templating base C, perhaps because the templated incorporation of G across C is particularly robust, and incorporation of the G monomer (across from any base) is also particularly efficient.2,3”

A single mutation would trigger a cascade of subsequent mutations and kinetic slowing.

“Our essential finding is that a single mutation would trigger a cascade of subsequent mutations and kinetic slowing.”

The mutation profile of an LNA-templated reaction resembled that of an RNA-templated reaction.

“We found that the mutation profile of an LNA-templated reaction resembled that of an RNA-templated reaction, implicating the A-form helix as the source of this difference.”

RNA systems exhibit higher mutation rates than comparable DNA systems.

“with an RNA system showing higher mutation rates than a comparable DNA system.20”

• cites [20] Leu K., 2011, Nucleic Acids Res. sourceNot checked yet

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Non-enzymatic extension has high mis-incorporation rates, between 7% and 26% per base on average across all bases, depending on the system.

“Mis-incorporation rates during non-enzymatic extension are quite high (error rate of 7-26% per base, averaged over all bases, depending on the system),18-20”

• cites [18] Rojas Stütz J. A., 2001, J. Am. Chem. Soc. sourceNot checked yet

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• cites [19] Kervio E., 2010, Proc. Natl. Acad. Sci. U.S.A. sourceNot checked yet

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• cites [20] Leu K., 2011, Nucleic Acids Res. sourceNot checked yet

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In a DNA model system using a 3′-amino primer and a nucleoside 5′-phosphorimidazolide monomer, mis-incorporation slowed copying of the next nucleotide by up to two orders of magnitude.

“In a particular DNA model system for non-enzymatic extension (primer terminated by a 3′-amino-2′,3′-dideoxynucleoside and the incoming monomer being a nucleoside 5′-phosphorimidazolide), mis-incorporation events slowed copying of the next nucleotide by up to two orders of magnitude.23”

• cites [23] Rajamani S., 2010, J. Am. Chem. Soc. sourceNot checked yet

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A single error would usually be followed by another error.

“Given our observation that the fidelity of incorporation immediately following a mismatch is very low, a single error would usually be followed by another error.”

The co-occurrence of multiple mutations suggests these systems may rapidly explore diverse areas of sequence space.

“The co-occurrence of multiple mutations suggests that these systems may be able to rapidly explore diverse areas of sequence space.”

Without errors, copying C in this context is a very high fidelity reaction (99.2%).

“Without errors, copying C in this context is a very high fidelity reaction (99.2%).20”

• cites [20] Leu K., 2011, Nucleic Acids Res. sourceNot checked yet

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Cooperativity among monomers enhances reaction efficiency, at least for G monomers.

“As seen for absolute rates, the reactions with the highest fidelities contained template ‘CC’ (1ca, 1ct), again suggesting cooperativity among monomers enhancing the reaction efficiency, at least for G monomers (Figure 7B, Supporting Table S4).”

Different sequence contexts suggest cooperativity among monomers affecting absolute rate (by up to two orders of magnitude) and fidelity.

“Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to two orders of magnitude) and fidelity.”

The fidelity of extension after a proper Watson-Crick base pair in the RNA system is 83%.

“For comparison, the fidelity of extension after a proper Watson-Crick base pair in this system is 83%.20”

• cites [20] Leu K., 2011, Nucleic Acids Res. sourceNot checked yet

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Non-enzymatic extension of RNA is relatively slow after a mismatch, to roughly the same extent as DNA.

“Although we did not examine extension from all possible matches and mismatches in the RNA system to calculate stalling factors, this illustrates that non-enzymatic extension of RNA is relatively slow after a mismatch, to a roughly similar extent as DNA.”

The ability of the incoming nucleotide to base pair in a Watson-Crick fashion with the template base is not very important if the primer terminus is mis-matched.

“This suggests that the ability of the incoming nucleotide to base pair in a Watson-Crick fashion with the template base is not very important if the primer terminus is mis-matched.”

The higher average fidelity on RNA template 5uu with imidazolides 4a-t was largely due to the relatively high fidelity (79%) with primer 2g (a G/U mismatch).

“A greater difference was seen on the RNA template 5uu (25% for 3a-t, 46% for 4a-t), largely due to the relatively high fidelity (79%) with primer 2g (a G/U mismatch) using 4a-t, which contrasts with the low fidelity after the same mismatch using 3a-t.”

Differences in magnitude of stalling factors (2-3-fold) observed for the DNA system here compared to prior work23 is likely due to the difference in sequence context.

“Since the leaving group had little effect, differences in magnitude of stalling factors (2-3-fold) observed for the DNA system here compared to prior work23 is likely due to the difference in sequence context.”

• cites [23] Rajamani S., 2010, J. Am. Chem. Soc. sourceNot checked yet

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Incorporation after a mismatch cannot be considered completely non-templated, because the absolute rate of incorporation can be 1-2 orders of magnitude higher with a template than without.

“Although the identity of the base is relatively unimportant, the incorporation cannot be considered completely non-templated, as the absolute rate of incorporation can be 1-2 orders of magnitude higher with a template than without (compare k′ in Table 2 vs. Table 5).”

• AssumptionRests on an assumption: The model reaction of activated monomers without template accurately estimates the rate of non-templated extension after a mismatch.

The phenomenon that an initial error triggers a cascade of extension problems appears to be general across nucleic acid systems.

“While each nucleic acid system has its own features, the phenomenon that an initial error triggers a cascade of extension problems appears to be general.”

• AssumptionRests on an assumption: Non-enzymatic polymerization may reflect the intrinsic chemistry underlying nucleic acid replication.

Surrounding sequence context or leaving group can affect stalling by 2-3-fold.

“Stalling factors previously observed in a completely different DNA sequence context with nucleoside 5′-phosphorimidazolides were slightly higher, in the range of 20-300,23 indicating that the surrounding sequence context (or leaving group; see below) can affect stalling by 2-3-fold.”

• cites [23] Rajamani S., 2010, J. Am. Chem. Soc. sourceNot checked yet

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The A-form helix is the source of the difference between LNA-templated and DNA-templated mutation profiles.

“We found that the mutation profile of an LNA-templated reaction resembled that of an RNA-templated reaction, implicating the A-form helix as the source of this difference.”

This study and similar studies that require observation of slow reaction rates are enabled by the amine nucleophile, which is more reactive than the hydroxyl of native DNA or RNA.

“This study, and similar studies that require observation of slow reaction rates, are enabled by the amine nucleophile, which is more reactive than the hydroxyl of native DNA or RNA.”

Simulations predict that the cycle of misincorporation and stalling would usually repeat several times, until the correct base is incorporated by chance, essentially allowing the duplex to close so templated polymerization may proceed.

“Our simulations predict that the cycle of misincorporation and stalling would usually repeat several times, until the correct base is incorporated by chance, essentially allowing the duplex to close so templated polymerization may proceed.”

If the size of a ribozyme were 30 bases, the simultaneous mutation of 5 consecutive bases would cause an evolutionary 'leap' through a relatively large region of sequence space with correspondingly large structural or phenotypic changes.

“For example, if the size of a ribozyme were 30 bases, the simultaneous mutation of 5 consecutive bases would cause an evolutionary ‘leap’ through a relatively large region of sequence space with correspondingly large structural or phenotypic changes.”

The nontemplated background rate establishes a basal rate of incorrect incorporation and limits the system from approaching very high fidelity through either mechanism.

“However, the nontemplated background rate establishes a basal rate of incorrect incorporation and limits the system from approaching very high fidelity through either mechanism.”

Primer extensions with amino-terminal primers give higher yields than those using hydroxy-terminated primers because the desired extension reaction competes more favorably with hydrolysis.

“Primer extensions with amino-terminal primers give higher yields than those using hydroxy-terminated primers because the desired extension reaction competes more favorably with hydrolysis.”

The local structure around the incorporation site can have large effects on extension rate.

“The local structure around the incorporation site can have large effects on extension rate.”

Simulations show that initial errors would tend to create relatively large stretches of consecutive mutations, suggesting that relatively large changes would occur at a single evolutionary step.

“Our simulations show that initial errors would tend to create relatively large stretches of consecutive mutations, suggesting that relatively large changes would occur at a single evolutionary step.”

This situation could combine the ability to store substantial genetic information with high evolvability.

“This situation could combine the ability to store substantial genetic information with high evolvability.”

There appears to be a cooperative effect, perhaps from stacking of adjacent monomers (particularly G), that enhances the rate of extension.

“As suggested by others,42 there appears to be a cooperative effect, perhaps from stacking of adjacent monomers (particularly G), that enhances the rate of extension.”

• cites [42] Kanavarioti A., 1993, J. Am. Chem. Soc. sourceNot checked yet

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