Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
DNA polymerases are complex molecular machines capable of replicating genetic material
using a template-driven process. While the copying function of these enzymes is well
established, their ability to perform untemplated DNA synthesis is less well characterized.
Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence
of a template. We use long-read nanopore sequencing, real-time fluorescence assays, and
atomic force microscopy to observe the synthesis and physical structure of pools of DNA
products derived from a diverse set of natural and engineered DNA polymerases across
varying temperatures and buffer compositions. We detail the features of the DNA fragments
generated, enrichment of select sequence motifs, and demonstrate that the sequence
composition of the synthesized DNA can be altered by modifying environmental conditions.
This work provides extensive data to better discern the process of untemplated DNA
polymerase activity and may support its potential repurposing as a technology for the guided
synthesis of DNA sequences on the kilobase-scale and beyond.

Introduction
Genetic information encoded in DNA sequences is a defining feature of all life on Earth. It
provides the instructions that cells require to synthesise and assemble their molecular
machinery, encodes signals to coordinate cellular processes, and is the substrate for the
process of evolution by natural selection. How biological information emerges de novo,
however, remains uncertain.

DNA polymerases (DNAPs) are typically viewed as molecular machines involved in
the high fidelity copying of genetic information through the replication of DNA using a template-
mediated process 1. However, it was shown in the 1960s that some DNAPs have the ability to
synthesise single-stranded DNA fragments in the absence of a pre-existing template 2–4. This
ab initio or “doodling” activity is widespread among polymerases 5 and has been observed in
RNA polymerases, notably T7 RNA polymerase 6, Qbeta RNA-dependent RNA polymerase 7
and 5TU RNA polymerase ribozyme 8. Doodling suggests a mechanism for how de novo
untemplated genetic information may be generated.

Investigations of this process have demonstrated that DNAPs have an intrinsic
capacity to doodle 9–12, that this process is sensitive to environmental factors such as
temperature and variations in buffer conditions 2,9,10,13, and that pools of synthesised DNA
molecules produced in the absence of template likely go through a multi-stage process of
semi-random synthesis and enrichment via the selection of sequences able to more efficiently
self-replicate 5,14–16. However, all such studies have been hampered by the inability to
adequately characterise the pools of DNA molecules generated. Recent advances in the
ability to read full-length DNA molecules using long-read sequencing have made it feasible to
generate near-complete sequence information from such vast pools, offering us insights into
the generative process.

In this work, we employ nanopore sequencing, real-time fluorescence assays, and
atomic force microscopy to provide the most comprehensive characterisation to date of
doodling kinetics, physical structure and sequence features of the full-length untemplated DNA
molecules synthesized (Figure 1a). We document the doodling function of a diverse array of
natural and engineered DNAPs, assess how temperature and buffer composition impacts the
nature of the sequences generated, and explore how this process may potentially be
manipulated through limiting deoxynucleotide triphosphate (dNTP) availability. We identify
considerably greater diversity in the features of the sequences generated than previously
reported, document the complexity of the DNA molecules synthesised, and demonstrate that
in select cases, modification of environmental conditions enables robust and directed changes
to the sequences produced. In addition to providing a rich data set for further characterizing
the nature of untemplated DNA synthesis, our findings also suggest a need to reconsider the
role of DNAP function in cellular biochemistry. Overall, the work offers a perspective on how
DNAPs may contribute to the genesis and evolution of new genetic information and may
provide the basis of future methods that repurpose DNAPs as programmable machines for
the synthesis of long DNA sequences.

Results
Single molecule analysis of doodled DNA
We began by focusing on Taq and Vent polymerases that have been shown to be capable of
untemplated DNA synthesis (Figure 1b) 9,12. Isothermal reactions were established using
standard buffers for each polymerase at 65°C and 74°C and run for 16 hours (Methods). We
then performed nanopore sequencing of each reaction to characterise the sequences present
in the pools of newly synthesized DNA fragments. To ensure that the reads containing
“doodled” products were accurate, we trimmed sequencing adapters from the ends of each
read, filtered out concatemer reads (namely those containing sequencing adapters within the
read itself that likely occur due to ligation of separate fragments during the preparation of the
sequencing libraries), and performed read filtering based on a median Q score ≥13 and length
>65 nt to remove sequences that might correspond to DNA fragments introduced when
preparing the sequencing libraries (i.e., adapter and barcode fragments) (Methods).

Analysis of the read length distributions showed substantive differences for each of the
polymerases (Figure 2, top sections of each panel). Taq displayed a bimodal distribution with
peaks at approximately 80 nt and 400 nt for reactions at 65°C, with a shift in the peak at 400
nt to 500 nt when the reaction temperature was increased to 74°C. This distribution also
displayed a much longer tail (Figure 2a,b), with over 20% of reads >1000 nt compared to only
4–7% at 65°C (Table 1). For both temperatures, similar concentrations of synthesised DNA
were measured (~1000 ng/µL). In contrast, Vent had only a single peak in the read length
distributions at ~80 nt, with a rapid exponential-type decay as read length increased that was
similar across temperatures and which resulted in only a very small fraction of reads being
>250 nt long (Figure 2c,d). The differences in the shape of the read length distributions
suggest that there may be factors (e.g., the formation of DNA secondary structures) that
hamper Vent’s ability to synthesise long DNA fragments. Additionally, ~10-fold less DNA was
produced at the lower temperature and only small fractions of reads were >1000 nt long (≤6%)
across both temperatures. This suggests Taq is more efficient at untemplated DNA synthesis
and able to produce longer DNA fragments than Vent, and that higher temperatures are more
conducive to the synthesis of longer untemplated DNA synthesis.

The nanopore sequencing additionally captured information regarding sequence
composition and characteristic repetitive patterns (i.e., sequence motifs) within each DNA
fragment. To characterize this dataset, we performed a number of linked analyses to generate
visualisations of the DNA pools where each analysed read is denoted by a vertical column
across all the plots (Figure 2, lower heatmaps). Analyses included assessing the base
composition (red heatmaps) for each read, the transition probabilities from one base to
another (blue heatmaps), and the sequence autocorrection to extract the scale (i.e.,
length/lag) of any repetitive sequences present (blue to yellow heatmaps). Each of these
analyses was performed for a subset of reads shorter and longer than 250 nt and the results
hierarchically clustered to ensure similar reads were grouped in the plots as vertical columns.
The top seven clusters were highlighted to simplify their classification (alternating dark and
light grey sections at the bottom of the panels). From these visualisations, it was possible to
assess distinctive features of each doodled DNA sequence and to make comparisons
between samples.

Using this approach, the Taq polymerase reactions at 65°C showed a majority of GT
repeats (61%) for reads <250 nt, in addition to less frequent AT repeats (13%), poly-A
sequences (4%), and random sequences (23%) (Figure 2a). In contrast, longer reads at this
temperature were nearly exclusively AT repeats (91%), with only a small fraction of GT repeats
and random sequences (9% in total). At 74°C, there was a distinctive shift in the sequence
composition, with smaller reads retaining the dominance of GT repeats (71%), and longer
reads demonstrating the emergence of GTATATAC repeats (93%) and a small fraction of
CATATATG (3%) repeats. The increased fraction of reads >1000 nt at 74°C, may relate to the
fact that these longer repeats (when found in multiples of 3 or more) can efficiently self-extend
by looping and base pairing of the complementary repeats and extension by Taq, using one
or more of the single stranded repeats as a template 5,14,15.

For the Vent polymerase (Figure 2c,d), there was again a dominance of repetitive GT
sequences (91–96%) and a small fraction of poly-A sequences (1–3%) for reads <250 nt
across both temperatures. However, for longer reads >250 nt, there were large differences
across temperatures as compared with Taq, with Vent reactions at 65°C showing only a small
number of repetitive GT and AT sequences, with reads instead being dominated by AATT
repeats (48%) and random sequences (37%) with a near even coverage of bases (Figure 2c).
This was substantially different to the AT repeat dominated Taq results (Figure 2a).
Interestingly, the Vent reactions at 74°C produced a small fraction of longer reads containing
GT repeats and random sequences (12% in total). However, the vast majority (88%) contained
CTATAG repeats (Figure 2d). This again, differed from the longer GTATATAC repeat
observed for the Taq reactions under similar conditions, and highlights potential biases in the
affinities these polymerases have for the addition of specific nucleotides. At 74°C, the longer
reads >250 nt demonstrated that Vent favours C → T, G → C, T → A and A → G or T, while
Taq has a strong preference for C → G, G → T, T → A and A → T transitions. Furthermore,
these preferences were influenced by temperature and appear to impact the efficiency with
which DNA synthesis occurs (different transitions dominate the analysis of the short and long
fragments).

Overall, these results were found to be robust for both polymerases tested and across
different temperatures, with similar patterns observed in biological replicates (Supplementary
Figures 1–4).

Assessing the capture of high molecular weight DNA
One of the major challenges when working with high molecular weight (HMW) DNA for
nanopore sequencing is the chance of shearing during the preparation of sequencing libraries.
This can lead to smaller than expected products. To assess whether this might be the case
for our results, which are generated from barcoded sequencing libraries to allow for the
assessment of many different polymerases and conditions within a single sequencing run, we
carried out an adapted sequencing protocol designed to help capture full length HMW DNA
molecules. This was performed for doodling reactions containing the Taq polymerase with the
Taq Standard buffer at 74°C (Methods).

Comparing the read length distributions of the barcoding and HMW protocols, we
observed a good reproducibility in the shape of the distributions across replicates
(Supplementary Figure 5). The most prominent difference was that there were virtually no
reads 65–250 nt long for the HMW protocol (<1%, Supplementary Figure 5b), whereas ~12%
for the barcoding protocol (Supplementary Figure 5a). This difference likely stems from the
barcoding requiring additional bead-based clean-up steps that could lead to minor shearing of
the DNA and the generation of smaller length fragments. In terms of longer reads, >99% were
longer than 250 nt and >35% longer than 1,000 nt for the HMW protocol, demonstrating its
ability to capture long reads. In contrast, the barcoding protocol saw an approximate 11%
fewer reads both >250 nt and >1,000 nt long compared to the HMW protocol. Importantly,
most of the very large fragments observed for the HMW protocol were between 250 nt and
2,000 nt, which were well captured by the barcoding protocol. Therefore, while some very long
sequences may not be captured by the barcoding approach, it is still able to provide insights
into the majority of types of molecule produced from a doodling reaction, and ensures
representative read length distributions.

Kinetics of doodling activity
Historically, the dynamics of doodling has principally been studied by time-sampling reactions
and visualizing DNA products by gel electrophoresis (Figure 3a) 17. To more precisely quantify
the doodling kinetics, we used a real-time fluorescence assay to track the formation of DNA
molecules synthesized under isothermal conditions. The experiments were run for 16 hours
at 74°C for both the Taq and Vent polymerases. In both cases, we found that doodling
occurred in two distinct stages (Figure 3b). Initially, a slow increase in synthesized DNA was
observed. Then, at around 30 min, a transition to a second phase occurred with an
approximately 3–3.5-fold increase in DNA synthesis rate. This rate was fixed (there was a
linear increase in DNA mass) for a further ~1.5 hours, after which the rate steadily decreased
until DNA synthesis stopped ~3 hours following the initiation of the reaction. This second stage
appeared to capture a saturation in the ability for DNA synthesis, likely due to the depletion of
free dNTPs or production of DNA products that cannot be further extended. This behavior was
consistent across replicates, with similar transition points and DNA synthesis rates (Figure
3b).

This two-stage process is consistent with a previously suggested mechanism of
untemplated DNA synthesis, whereby an initial stage allows for the generation of short random
DNA sequences that may subsequently by chance discover repetitive sequences able to more
efficiently self-replicate in a second stage 5,14,15 (mentioned previously). These results
therefore further support the role of self-replicating sequences in the generation of new genetic
material during the doodling process.

Influence of environmental factors
It has been demonstrated that environmental factors such as temperature and buffer
composition can influence the length and sequence composition of doodled DNA 2,9,10,13. While
we have shown the important role that temperature plays in determining sequence
composition (Figure 4), to further characterize this effect, we studied the doodling activity of
Taq polymerase across a range of buffers. These included a minimal buffer with varying pH
(8.2 and 9.5) and a standard Taq buffer including excessive amounts of MgCl2, which is known
to stabilize the annealing of primers to incorrect template sites when used in PCR reactions
and thereby decreasing the specificity of the amplification process.

Similar to previous experiments, the results were shown to be highly reproducible for
many of the conditions, with similar patterns observed across experimental replicates
(Supplementary Figures 6–9). The main exceptions were Taq at 65°C in Taq standard buffer
supplemented with 25 mM MgCl2 and Taq at 74°C in minimal buffer at pH 8.2. Replicates
under these conditions resulted in similar qualitative features for the read length distributions,
but quantitative differences in the ratio of short and long fragments. A comparison of the
sequence composition and repetitive motifs across different conditions showed that in most
cases the same patterns were maintained. The only notable differences were for Taq at 65°C
when using a minimal buffer at both pH 8.2 and 9.5, which displayed a slight decrease (>2-
fold) in the fraction of GC repeats for reads <250 nt (Figure 4b,c), and the more complex
sequence repeats produced by Taq at 74°C in the minimal buffer at pH 8.2 (Figure 4a), which
for one of the replicates (replicate 1) also contained longer repetitive sequences at smaller
(<250 nt) read lengths (Supplementary Figure 9).

The most notable differences observed were in the amount of DNA synthesized and
the fraction of long (>1000 nt) DNA sequences produced (Table 1). Most of the reactions saw
a decrease in the amount of DNA synthesized (apart from those at 65°C in the Taq Standard
buffer with MgCl2), with the minimal buffer leading to an ~10-fold decrease at 74°C and ~100-
fold decrease at 65°C. Smaller drops in the percentage of long reads >1000 nt were also seen,
as well as differences in the shape of the read length distributions. This suggests underlying
changes in the DNA synthesis process. Specifically, reactions that used the minimal buffer at
65°C with a pH of 8.2 or 9.5 resulted in far fewer small DNA fragments <250 bp, and read
length distributions with a symmetric Gaussian-like shape (Figure 4b,c). This would be
indicative of a generative process whereby all fragments in the pool have similar rates of
synthesis, but where there is some stochasticity in the process to cause variation at the pool
level. Interestingly, these reactions were dominated by reads containing AT repeats, with little
diversity in the repetitive motifs present (especially for longer reads >250 bp). Given the
amplification of different motifs is likely to occur at different rates, having only a single
dominant motif may account for the simpler read length distribution in these specific conditions
(i.e., it imposes specific constraints on the typical maximum length that can be achieved).
Together, these results highlight the role that environmental factors play in biasing the
sequence composition and affecting the synthesis efficiency of the doodling process.

Impact of cycling thermal conditions
All experiments so far were carried out under isothermal conditions. As the amplification of the
repetitive sequences would require binding and unbinding of the ssDNA molecules to proceed
efficiently, we wondered whether thermal cycling might enhance this process. To test this
hypothesis, doodling reactions for both Taq and Vent were established where the temperature
was cycled 9 times between 65°C for 20 mins and 74°C for 20 mins, for a total reaction time
of 6 hours. DNA from these reactions was then nanopore sequenced and analyzed in a similar
way as for the isothermal reactions.

Counterintuitively, we found that for both Taq and Vent, temperature cycling virtually
eradicated the occurrence of repetitive sequences, with only a small fraction (<2.5%) showing
a repetitive sequence motif (Supplementary Figure 10a). Interestingly, at least an order of
magnitude less DNA was produced in these reactions compared to the normal isothermal
doodling reactions (<12% compared to Taq and Vent at 74°C, and 1% compared to Vent at
65°C), and there was a near uniform transition from one base to another in the sequences
produced (Supplementary Figure 10b). More detailed analysis showed that all samples were
dominated by sequences that were a mixture of contaminants, mostly from the Escherichia
coli genome (Supplementary Data 1). Partial sequences were commonly observed for the
uncharacterized ydfO, ybcW and ylcI genes related to the DLP12 and Qin prophage. DNA
fragments containing these sequences were likely carried through from the purification of the
enzymes by the manufacturers and the cycling of temperature during the reactions may have
allowed for non-specific binding and extension to create the small number of chimeric
contaminant sequence seen. While commercial providers do guarantee only trace amounts of
such contaminants are present in their products (≤1 genome per µL from quality control
assessments using qPCR by the commercial providers of the reagents used in this study), the
sensitivity of the nanopore sequencing enables them to be picked up when virtually no
doodling occurs.

To assess whether these contaminants might confound the results of other doodling
experiments, we analyzed the E. coli BL21(DE3) genome to see whether the repeats we
observed in our doodling reactions that are able to self-amplify could have been generated by
template-mediated synthesis from genomic contaminants (Methods). Only a single
occurrence of a repeated GATATC motif was found. This makes it highly unlikely that genomic
DNA contaminants from the enzyme purifications are the cause of the diverse and highly
repetitive sequences that dominate the doodling reactions. Furthermore, the very small
amounts of contaminants in the isothermal doodling reactions, suggests that amplification of
these products is unable to proceed efficiently, unlike the doodling process.

Physical structure of doodled DNA pools
Sequencing of doodled DNA products provides some insight into the composition of the DNA
produced, but is unable to capture the physical structures these molecules produce
individually and through interactions with other molecules within the pool. To assess these
features, we performed atomic force microscopy (AFM) for the Taq polymerase reactions. We
used a high-speed contact mode AFM approach, which allowed us to image large-scale
structures (areas of 1.3 µm2 per image) at a sub-nanometer resolution (Figure 5a; Methods).
From the AFM images we were able to extract 788 molecules at sufficient contrast to
accurately evaluate their structure. It was immediately evident that a large proportion of the
molecules displayed a clear branching structure (Figure 5b) with 23% of the molecules
containing one or more branches. In addition, there was a steep drop off in the number of
branches per molecule and a maximum of 8 branches seen in a few cases (Figure 5c). Given
that ssDNA molecules are synthesized, these branches likely occur either at common repeat
sequences found across different molecules or by a single molecule folding back on itself at
repetitive, palindromic sequences to create a tight hairpin with a non-binding end that is
observed as a separated branch. Closer inspection of some of these imaged molecules
showed increased peaks in height at points where branches occurred (Figure 5b, white
arrows). This suggests that these regions may be double stranded, supporting the idea of
base pairing between separate ssDNA molecules, or through the formation of hairpins within
a single ssDNA molecule.

The ability to view the DNA molecules allowed us to calculate physical lengths that
could then be converted into numbers of nucleotides using the fact that a single nucleotide is
0.34 nm long 18 (Figure 5d). We calculated median DNA molecule lengths of 518 nt and 552
nt from the AFM and nanopore sequencing data, respectively, for the same Taq doodling
reactions at 74°C with the Taq Standard buffer. The longest molecule we imaged was
estimated to be 2,769 nt long. The small difference of ~6% in the median molecule lengths
lends further support to the nanopore sequencing recovering accurate lengths of the
molecules present in these reactions.

As well as providing support for the accuracy of the sequencing data, the branched
structure of many of the doodled molecules could also provide an explanation for the very long
DNA molecules (>10,000 nt) inferred from the electrophoresis gels (Figure 1b and Figure
3a), but not observed at such high levels in the sequencing data or via AFM. The reason could
be that rather than there being very long ssDNA molecules present, instead the ability for the
ssDNA sequences to base pair with each other through the common repetitive motifs would
enable the creation of very large multi-molecule structures that would run much slower through
a gel than a single-molecule counterpart. This would confound the typical interpretation of
these gels and further highlights the importance of performing complementary measurements
like sequencing and AFM to better understand poorly understood biological processes.

Doodling activity of other DNA polymerases
Given the distinct sequence compositions of the doodled products derived using Taq and Vent
polymerases, it appears likely that other DNAPs may have unique biases in the untemplated
sequences they generate. To further examine this, we considered two other DNAPs: Vent
exo– to determine whether mutations in Vent that remove its proofreading exonuclease activity
impact doodling, and Therminator as it was originally sourced from a distantly related species
(Thermococcus species 9°N-7) and is known to incorporate modified substrates. In addition,
we also studied two engineered DNAPs, RT521K and 3A10 19,20. RT521K is an extensively
engineered variant of Tgo polymerase 19 and 3A10 is a chimera of Tth and Taq polymerase
generated by molecular breeding and compartmentalized self-replication (CSR) selection 20.
We carried out 16 hour isothermal reactions and performed nanopore sequencing to
characterize the sequences of the doodled DNA products (Methods).

Highly reproducible results were seen for all four polymerases, with read length
distributions displaying an exponential decay for longer read lengths, similar to previous
experiments with Vent (Supplementary Figures 11–14). For Vent exo–, we found large
fractions of reads containing GT repeats (93% of reads <250 nt), a lack of the more complex
AATT repeat sequences seen with standard Vent polymerase, and an increase in the fraction
of reads with near-random sequences (65% of reads >250 nt) (Figure 6a). Therminator
displayed similar features, but contained a small number of longer reads (>250 nt) with more
complex sequence repeats, including CTATAG, GTATATAC and AATT (3–8% total across
replicates) (Figure 6b). For the engineered polymerases, we found that the short reads of
RT521K were once again dominated by GT repeats, but virtually all longer reads >250 nt had
random-like sequences (97%) (Figure 6c). This was evident from the near-equal fraction of
bases and transition probabilities between bases. The 3A10 polymerase showed a similar
dominance of random-like sequences, although to a lesser extent than RT521K (Figure 6d).
When MnCl2 and MgCl2 were added to the buffers for the RT521K and 3A10 reactions,
respectively, the probability of a shift to more random doodling became greater. For MnCl2,
this is likely due to the additive promoting the misincorporation of nucleotides and destabilizing
existing biases in base transitions 21. The most distinctive difference of these studied
polymerases compared to Taq and Vent was the heavily reduced overall quantity of DNA
synthesized per reaction (<4 ng/µL in all cases) and a lower fraction of reads >250 nt long
(Table 1).

Analysis of DNA polymerase transition frequencies
The polymerases considered so far span both Family A and B, which are known to have
structural and functional differences that affect their mutational biases 22. To assess whether
these biases might underpin some of the favored transitions seen in the doodled DNA
products, we generated averaged base transition probabilities for each DNAP and condition,
grouping the polymerases by Family or classifying them as an engineered variant (Figure 7a).
From these transition matrices, it was clear to see common Family related biases. Specifically,
in the doodled DNA products, all Family A DNAPs had a strong preference for A → T, T → A
and G → C transitions, and a weak preference for C → A transitions. Furthermore, a change
in temperature to 74°C leads to the emergence of an additional strong preference for C → G
transitions. Family B DNAPs show a different pattern, with a strong preference for G → T, T
→ G transitions and more even transitions from A and C nucleotides to all others at 65°C, and
the emergence of a strong C → T transition at 74°C for Vent. These trends are mirrored for
the 3A10 engineered polymerase, which is a chimeric variant made from two Family A DNAPs
(Tth and Taq). Interestingly, the RT521K polymerase, which is from Family B and is the most
heavily engineered of those studied, showed fewer overall biases. This leads to near even
transition probabilities between all bases in the doodled DNA products and is also evident in
the fewer repetitive motifs seen in the sequence pools.

It is known that most DNAPs have a much higher propensity for base substitutions
being transitions (A ↔︎ G or C ↔︎ T) as opposed to transversions (A ↔︎ T, C ↔︎ G, A ↔︎ C or T
↔︎ G). In particular, while A ↔︎ T and C ↔︎ G transitions in the doodled DNA products are
prominent across all DNAPs (Figure 7a), these types of transversion during base substitutions
are typically rare being <10% and <5%, respectively, for many DNAPs 22. This suggests that
the structural constraints for doodling activity are very different to those affecting DNA copying
fidelity and that these biases can be shaped through DNAP engineering.

Analysis of repetitive sequence motifs
It is possible for a single DNA molecule to contain numerous different types of repetitive
sequence throughout its length. These would not be captured by our previous autocorrelation
analysis or through the calculation of transition frequencies (Figures 2, 4 and 6). To better
characterize the full range of repetitive motifs present in the sequencing data, we performed
an analysis in which we searched for a set of common motifs found across all the polymerases
and conditions assayed. We then calculated the fraction of reads in which these repetitive
motifs were found (Figure 7b). It should be noted that this analysis is more sensitive than the
autocorrelation analysis to single base differences, with exact motif sequences matches
required and a minimum number of repeats (at least 7 consecutive repeats of 1–3 nt motifs, 6
consecutive repeats of 4 nt motifs, and 4 consecutive repeats of 6–8 nt motifs).

From this analysis, we observed clear differences in the patterns of repetitive motif
present for different polymerases, temperatures, and buffer compositions (Figure 7b). One of
the most striking features was the high frequency of AT repeats for Taq, and long T-
homopolymers and TG repeats for the other polymerases, as well as the synthesis of reads
with few repetitive motifs for RT521K and Taq at 74°C with a minimal buffer at pH 8.2. More
generally for both Taq and Vent, repeats of a longer length (>4 nt) were more frequently seen
at 74°C compared to 65°C, where shorter 1 or 2 nt long repeats dominated. Poly-G repeats
were rare for all polymerases and CG repeats for all polymerases except Taq. However, the
addition of MgCl2 at 65°C saw the complete loss of CG repeats for Taq. Very specific repeats
also emerged for some of the polymerases under certain conditions: AATT and GGT repeats
were uniquely seen for Vent at 65°C, CTATAG repeats for Vent at 74°C, and GTATAC and
GTATATAC repeats for Taq at 74°C in a Taq Standard buffer. Furthermore, there was a near
complete breakdown of GTATATAC repeats for Taq at 74°C when a minimal buffer at pH 8.2
was used, highlighting how sensitive the synthesis processes are to environmental conditions.

Guiding sequence composition using dNTP availability
Having verified that experimental conditions like temperature and buffer composition could
influence doodling behavior, we next sought to determine whether availability of dNTPs could
be used to guide the sequence of doodled DNA, providing a potential means by which some
degree of control could be obtained over the sequence composition of the generated DNA
fragments.

In the first instance, we carried out a set of additional real-time fluorescence
experiments to determine whether the availability of only single types of dNTP or pairs of dNTP
may influence the kinetics of doodling for the Taq and Vent polymerases (Figure 8a). We
found that for single types of dNTP virtually no doodling was observed for both polymerases.
In contrast, when two different types of dNTP were present, consistent normalized DNA
synthesis curves were obtained that closely matched reactions in which all dNTPs were
present for both Taq and Vent (Figure 8a). However, unlike reactions in which all dNTPs were
present, most reactions with limited types of dNTPs synthesized much less DNA (<4 ng/µL
compared to >100 ng/µL) as compared with the situation when all dNTPs were present
(Supplementary Table 1). This is unsurprising given the biases previously observed in the
ability of DNAPs to incorporate specific dNTPs into a DNA molecule when a base of a
particular type is present at the 3’ end.

To determine whether the limited dNTP availability affects the composition of the
sequences produced, we performed nanopore sequencing on each reaction, as well as a
further reaction containing three dNTP types (TCG). Analysis of the sequence motifs showed
a bias for poly-T sequences and repeats of AT, TG and GTATATAC for all the single, double,
and triple dNTP reactions, except for when only adenine and thymine were present (Figure
8b). In that case, we found as expected that sequences were nearly entirely composed of
those bases (87% of reads <250 nt and 99% of reads >250 nt). Interestingly, short and long
reads were dominated by A4T4 repeats in addition to A5T5 repeats at a lower frequency (73%
and 26%, respectively, across all reads >65 nt long) (Figure 8c). While qualitative features of
these repetitive sequence motifs were consistent across experimental replicates
(Supplementary Figure 15), much greater variation in the absolute factions of specific motifs
was seen compared to prior doodling reactions. Specifically, the A4T4 and A5T5 motifs that
dominated both reactions saw drops of 16% and 10%, respectively across replicates. This
suggests that these reactions may be more sensitive to minor differences in the setup of the
reactions and external contaminants. Unlike all other reactions with limited combinations of
dNTPs, where the majority of reads were <1000 nt (>80%), when only adenine and thymine
were present >50% reads were long with a length >1000 nt. This suggests that these
sequence motifs are more efficiently synthesized, likely due to their ability to self-replicate if
the single-stranded DNA encoding them bends and base pairs with itself, allowing for standard
extension and replication. Overall, these results demonstrate the potential to modify the
composition of doodled DNA, in some cases by tuning dNTP availability, and may offer a
means for the synthesis of long DNA fragments where the specific sequence is less
consequential, but where statistical composition must be maintained.

It is unclear how DNA synthesis with bases not included in the reaction mix can take
place, but could be due to contaminants in the individual dNTP mixes or polymerase
preparations used (the commercial provider of the reagents used here guarantee ≥99% pure
dNTPs determined by HPLC) as well as the high sensitivity of the nanopore sequencing. A
less likely cause could also be the nanopore sequencing library preparation. However, such
sequences have not been seen previously and do not match any of the barcode or adapter
sequences used. If inclusion of these bases was due to contamination in the doodling
reactions, their limited availability may account for the small amounts of DNA synthesized in
these reactions (Supplementary Table 1).

Another possibility for the observed guanine and cytosine bases in the sequencing
data is the high and sustained temperature of the doodling reactions, which could hydrolytic
deamination of adenine to inosine. Given that the basecaller we use is not trained to detect
inosine bases, if encountered, they could be miscalled as guanine or cytosine bases. To
investigate this, we synthesized a pair of oligos (dIx3_F and dIx3_R; Supplementary Data 2)
with complementary base-pairing flanks around a 3 bp central region that contained a repeat
of inosine bases on the forward strand, and thymine on the reverse strand to mimic
deamination of adenine bases (Supplementary Figure 16a). These oligos were annealed to
create dsDNA molecules and nanopore sequenced. Raw sequencing data was then
basecalled, reads for each oligo separated, and the central region between the common
flanking sequences extracted (Methods).

Reads of the forward oligo containing the inosine bases showed clear miscalling, with
the majority of the central region given a G/C-based sequence, with CGC being the most
common (32% in total) (Supplementary Figure 16b). In contrast, the reverse strand saw
accurate calling of the three thymine bases (72% in total) (Supplementary Figure 16c). This
suggests that some of the guanine and cytosine bases seen in our nanopore data may arise
from miscalling of deaminated adenine bases. However, given the results for when only
adenine and thymine are present, where >95% of bases across all reads matched these bases
(Figure 8b), it is unlikely that deamination is the major contributor to the unexpected guanine
and cytosine bases seen in the sequencing data for the other doodling reactions with limited
types of dNTP.

Seeding reactions with self-replicating oligos to guide doodling activity
Having shown that doodling could be guided through changes in reaction conditions (e.g.,
temperature and available dNTPs), we finally wondered whether the amplification step could
also be harnessed to sense changes in environmental conditions by seeding reactions with
ssDNA oligos able to self-amplify. In particular, we took inspiration from differential sensing
approaches that mimic our olfactory system and which have also been implemented in
synthetic biology using de novo designed protein barrels 23.

To examine this, we developed a small panel of 6 ssDNA oligo sequence designs that
contained repeat motifs allowing for self-amplification via either inter or intra DNA molecule
binding (Figure 9a; Methods). We considered repeat motifs of 2, 4 and 6 nt long and for the
6 nt motifs, elaborated on the CTATAG repeat seen in previous doodling reactions, altering
the order of the T/A nucleotides in 4 different ways to create the motifs: CTATAG, TCATGA,
CTTAAG, CAATTG. These motifs, should have similar binding energies, but we hypothesised
may have differing amplification efficiencies given only one of them had been seen during
previous doodling reactions. Because the seed sequences would be provided to the reaction
at the start, we also expected a stronger signal in the amplified pools and more consist and
reproducible outcomes.

Isothermal doodling reactions were setup with equimolar amounts of each ssDNA seed
oligo (0.2 µM total for all oligos combined) for the Vent DNAP. As a simple test, we exposed
the reactions to 65°C and 74°C to see if a different fingerprint in the final DNA pool composition
would be seen. As expected, we found major differences in the fraction that each oligo was
present and saw high reproducibility across our replicates, with only very minor differences in
the pool compositions (Figure 9b). Interestingly, for the 6 nt motifs that all contained an
identical nucleotide composition, there were substantial differences in the relative changes
with >18-fold increase for the CTATAG motif, an approximately 50% reduction for the
TCATGA and CAATTG motifs, and a near 100% reduction in the CTTAAG motif (Figure 9c).
This suggests that the amplification process is highly sensitive to minor differences in the
specific ordering of nucleotides of the seed oligos, a feature that could potentially be exploited
to aid discrimination of other environmental factors that might affect aspects of DNA folding
and binding to other DNA molecules and the DNAP.

These results demonstrate the ability to exploit the differential amplification of ssDNA
molecules during isothermal doodling reactions for encoding features of the reaction
environment into the composition of the pool of DNA molecules that are synthesised,
suggesting yet a further application of this process beyond de novo DNA synthesis.

Methods
Standard in vitro reactions
All doodling reactions were performed in thin wall PCR tubes or 96-well PCR plates, using
reaction volumes of 100 µL with 200 µM of dNTPs (New England Biolabs, N0447S).
Reactions were prepared on ice in a PCR hood before being transferred to a preheated
thermocycler (Bio-Rad, C1000). Nuclease-free water (Omega Biotek, PD092) was used to
prepare all reactions. Reactions were run for 16 hours at 64°C or 75°C, unless stated
otherwise. Taq reactions were performed in a 1X Taq Standard Buffer (New England Biolabs,
B9014) containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, and 5 units of polymerase
(New England Biolabs M0273). Vent reactions were performed in 1X Thermopol buffer (New
England Biolabs, B9004) containing 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM
MgSO4, 0.1% Triton X-100, and 2 units of polymerase (New England Biolabs M0254). Vent
exo– (New England Biolabs, M0257) and Therminator (New England Biolabs, M0261)
polymerase reactions were set up similarly to those for Vent. RT521K polymerase reactions
were performed in 1X Thermopol buffer supplemented with 25 Mm MnCl2. 3A10 polymerase
reactions were performed in 1X Taq standard buffer supplemented with 25 mM MgCl2. For
RT521K and 3A19 polymerase reaction, 1 µL (5 U/µL) of polymerase was used. 1X minimal
buffer was formulated as 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2 and adjusted to pH 8.2
at 25°C or pH 9.5 at 25°C.

In vitro reactions with temperature switching
Temperature switching reactions were performed in thin wall PCR tubes, using reaction
volumes of 100 µL with 200 µM of dNTPs (New England Biolabs, N0447S). Reactions were
prepared on ice in a PCR hood before being transferred to a preheated thermocycler (Bio-
Rad, C1000). Nuclease-free water (Omega Biotek, PD092) was used to prepare all reactions.
Duplicate reactions were performed over a total of 6 hours, consisting of 9 cycles of 64°C for
20 minutes followed by 75°C for 20 minutes. Taq reactions were performed in a 1X Taq
Standard Buffer (New England Biolabs, B9014), and 5 units of polymerase (New England
Biolabs, M0273). Vent reactions were performed in 1X Thermopol buffer (New England
Biolabs, B9004), and 2 units of polymerase (New England Biolabs, M0254). Prior to
sequencing, PCR products were purified using Beckman Coulter AMPure XP beads (Fisher
scientific, A63880) at a bead-to-sample ratio of 1.8:1.

In vitro reactions with seed oligos
Isothermal amplification reactions were performed in thin-walled PCR tubes with 100 µL total
reaction volumes. Reaction mixtures contained 200 µM dNTPs (New England Biolabs,
N0447S), 0.2 µM of a single seed oligo (seed_ATAT_5, seed_AATT_5, seed_CTATAG_3,
seed_TCATGA_3, seed_CTTAAG_3, seed_CAATTG_3; Supplementary Data 2), 1X
ThermoPol buffer (New England Biolabs, B9004), and 2 µL of Vent DNA polymerase (New
England Biolabs, M0254). Nuclease-free water (Omega Biotek, PD092) was used in all
reactions. Reactions were assembled on ice in a PCR hood and incubated isothermally at
65°C and 74°C for 16 hours. A no-seed oligo control was included. For pooled seed oligo
reactions, six seed oligos were combined to a final concentration of 0.6 µM (0.1 µM each).
Reactions were performed in 100 µL volumes containing 200 µM dNTPs (New England
Biolabs, N0447S), 1X ThermoPol buffer (New England Biolabs, B9004), and 2 µL of Vent DNA
polymerase (New England Biolabs, M0254). Nuclease-free water (Omega Biotek, PD092) was
added to a final volume of 100 µL. Reactions were incubated isothermally at 65°C and 74°C
for 16 hours and were performed in duplicate. Prior to sequencing, PCR products were purified
using Beckman Coulter AMPure XP beads (Fisher scientific, A63880) at a bead-to-sample
ratio of 1.8:1.

Gel electrophoresis
Gel electrophoresis was performed for DNA length quantification of reaction products. Gels
were prepared using 1% agarose in 30–50 mL of 1X TAE running buffer (40 mM Tris-acetate,
1 mM EDTA). Gels were stained by adding 1X nucleic acid stain Gel Green (Biotium, 41005)
to the gel solution prior to casting. 50 µL of sample was loaded into each lane of the gel, using
10 µL purple loading dye (New England Biolabs, B7025S). The DNA 1 kb+ ladder (New
England Biolabs, N3200) was used as a reference. Gels were run at 80V for 30–60 minutes
(Bio-Rad, PowerPac Basic).

Nanopore DNA sequencing
For most samples, DNA was prepared for nanopore sequencing following the ligation
sequencing of amplicons protocol using Oxford Nanopore Technologies (ONT) kit SQK-
LSK110 directly, or the native barcoding protocol using ONT kits SQK-LSK110 and EXP-
NBD104. DNA quantification was performed using a Qubit dsDNA broad range DNA
quantification kit and a Qubit 4 fluorometer (Invitrogen, Q33238). For barcoding, equal
volumes of each barcoded sample were pooled and 6–60 ng of DNA was loaded into each
flow cell (ONT, FLO-MIN106). Sequencing was performed using a MinION Mk1B device and
the MinKNOW software version 21.11.7. DNA samples for temperature switching and seed
oligo experiments were prepared for nanopore sequencing using the Native Barcoding Kit 24
V14 (ONT, SQK-NBD114.24) and loaded onto FLO-MIN114 flow cells. For barcoding, equal
volumes of each barcoded sample were pooled and 8–112 ng of DNA was loaded onto the
flow cell. For seed oligo experiments, individual seed oligos and pooled oligos, incubated at
either 65°C or 74°C, were loaded onto the flow cell at a final pooled DNA concentration of 566
ng. The sequencing library preparation for the high molecular weight samples used an
adapted protocol for the Ligation Sequencing DNA V14 kit (ONT, SQK-LSK114), with libraries
loaded onto FLO-MIN114 flow cells. Specifically, only a single sample was sequenced per
flow cell to reduce the number of clean up steps required before sequencing (i.e., clean up
steps after barcode ligations), and all steps were carried out using wide bore pipette tips and
performed very slowly to minimize the chance of DNA shearing throughout.

Real-time tracking of DNA synthesis using fluorescence
A real-time PCR machine was used to monitor using fluorescence the synthesis of doodled
DNA products during a reaction. For the Taq polymerase, 100 µL reactions were prepared on
ice in 96-well plates containing 84 µL nuclease free water (Omega Biotek, PD092), 10 µL of
10X Taq standard buffer (New England Biolabs, B9014), 2 µL of 10mM dNTPs (complete
mixtures of all dNTPs, New England Biolabs, N0447S; or combinations of single types of dNTP
supplied as a separate set, New England Biolabs, N0446S), 1 µL Taq polymerase (2 units
final concentration), 2 µL ROX solution (ThermoFisher Scientific, R1371), and 1 µL SYBR
Green dye (ThermoFisher Scientific, S7563). For the Vent polymerase, identical reactions
were prepared, however, the 10X Taq standard buffer was replaced with a 10X Thermopol
buffer (New England Biolabs, B9004). Assembled reactions were then run in a preheated
qPCR machine (Agilent Technologies, Stratagene MXP3005P) with a fixed temperature of
74°C and a cycle time of 5 min for a total of 16 hours. Measurements of fluorescence (SYBR
Green and ROX) were taken each cycle.

Atomic force microscopy
AFM was performed on Taq DNA samples generated using 100 µL reactions. Reactions were
assembled in batch to minimize pipetting error and contained 1X Standard Taq Buffer (New
England Biolabs, B9014), 200 µM dNTPs (New England Biolabs, N0447S), 5 units of Taq
polymerase (New England Biolabs, M0273), and nuclease-free water (Invitrogen). Reactions
were incubated at 74°C for 16 hours and performed in triplicate. DNA was subsequently
purified using AMPure XP beads (Beckman Coulter, A63880) at a 1.8:1 bead-to-sample ratio
and eluted in 40 µL nuclease-free water. Samples were diluted in Tris-MgCl2 deposition buffer
(10 mM Tris-HCl, 5 mM MgCl2, pH 7.6), freshly prepared and heated to 80°C for 1 hour prior
to use, to a final concentration of 0.02 µg/mL for AFM imaging. To enable high-resolution
imaging of individual DNA molecules, the samples were deposited onto an atomically flat
freshly cleaved mica substrate. This surface facilitates optimal contrast and resolution for
visualizing double-stranded DNA, which has a diameter of approximately 2 nm. A 1 µL DNA
sample was applied to the freshly-cleaved mica surface and incubated for 1.5 minutes,
followed by three washes with 200 µL Milli-Q water. The samples were then dried using a
gentle stream of dry air to remove any water droplets. Mica discs were then baked at 120°C
for 20 minutes, cooled to 40°C (covered dry bath followed by covered bead bath). Finally, the
prepared mica substrates were affixed to SEM stubs and imaged using a high-speed atomic
force microscope (HS-AFM).

The AFM used for this study was a custom contact-mode HS-AFM developed at the
University of Bristol, UK 54. It is able to generate two 1-megapixel topographic maps per
second with a height resolution of ±15 picometres and has an adjustable scan size of 500 x
500 nm to 30 µm x 15 µm per image. Previous versions of this instrument have demonstrated
the feasibility of using contact-mode HS-AFMs as genomic diagnostic tools 55. These studies
have shown that the high-spatial resolution and rapid imaging capabilities can be used to
generate large (>1.5 million molecule) data sets in practical timescales and at competitive
price points per test 56. Here, we used the high throughput of the HS-AFM to survey the high
molecular weight samples of doodled DNA and measure key characteristics such as the
backbone length and number of branches of over 700 individual DNA molecules.

For analysis, it was necessary to adapt existing image analysis algorithms for DNA
detection and measurement because many of the doodled DNA molecules featured one or
more branches. Typical DNA analysis routines cannot distinguish the difference between a
single branching DNA molecule and multiple overlaid molecules. For this study, a semi-
automated approach was implemented. In brief, each raw AFM image was flattened using a
line-by-line 3rd order polynomial, and a threshold used to identify DNA molecules. After this
step the operator inspected each detected DNA molecule and in the case of any strands with
branches, they identified the branching locations by manually selecting the junctions with the
mouse pointer. The backbone and any branches were then traced using a spline fit to the
skeletonized threshold image. This enabled each molecule to be defined not just in terms of
its backbone length but also by the number and length of any branches from this backbone.

Sequencing data analysis
Raw sequencing data in FAST5 format was basecalled using guppy version 6.3.8 and the
configuration file ‘dna_r9.4.1_450bps_hac.cfg’. The resultant FASTQ files were then cleaned
by trimming any adapter or barcode sequences found at the start or end of each read and
reads with barcodes found internally were removed. Unless otherwise stated, we also filtered
reads based on length >65 nt and Q-score ≥13 to enable more accurate inference of statistical
and repetitive features of the sequences. Autocorrelation analyses were performed by
calculating a discrete and normalized string-based autocorrelation function r(k), where k is the
lag (shift in sequence). We assume that only perfectly matching elements (bases) are included
in the calculation with the final value normalized by sequence length to account for the finite
nature of the input sequence. This was implemented as a custom function in Julia.

Analysis of contaminant sequences
To analyze the source of contaminant sequences in the temperature switching experiments,
we performed a detailed annotation of a random selection of reads using the pLannotate tool
57. Selected sequences were manually extracted from the processed FASTQ file for replicate
1 of the temperature switching experiment and annotations generated using the pLannotate
web interface. CSV files containing the annotations for each read were downloaded and then
compiled into a single spreadsheet (Supplementary Data S1).

Analysis of E. coli genome for repetitive sequence motifs
Analysis of the E. coli genome was performed using the complete sequence of the BL21(DE3)
strain (GenBank: CP001509.3). Julia scripts were used to search for occurrences of repetitive
sequence motifs that were able to self-replicate and which were seen in the doodling reactions.
The proportion of the genome encoding them calculated as (n × m) / g, where n and m are the
number of occurrences and length of the motif, respectively, and g is the total length of the E.
coli BL21(DE3) genome sequence (4,558,953 bp). We specifically searched for the following
motifs: (CTATAG)2, (CTATATAG)2, (CATATG)2, (CATATATG)2, (GTATAC)2, (GTATATAC)2,
(GATATC)2 and (GATATATC)2. Only perfect matches were considered.

General computational analysis tools
All analysis scripts were developed and run using Julia version 1.10.2 and the following
packages: BioAlignments version 3.1.0, BioSequences version 3.1.6, Clustering version
0.15.7, CSV version 0.10.14, DataFrames version 1.6.1, Dates version 1.10.0, Distances
version 0.10.11, FASTX version 2.1.4, Glob version 1.3.1, LinearAlgebra version 1.10.0,
Random version 1.10.0, and Statistics version 1.10.0. Plots were generated using Julia and
the CairoMakie version 0.12.3 package and final figures composited in Affinity Designer
version 2.5.3.

Statistics and reproducibility
Sample sizes were chosen to match typical practices in the field (i.e., biological duplicates for
DNA sequencing data). No data were excluded from the analyses. The experiments were not
randomized. The Investigators were not blinded to allocation during experiments and outcome
assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript from Castle et al looks at the untemplated DNA synthesis activity of several DNA polymerases. In particular,\nthey use long read synthesis to analyze the sequence composition resulting from the untemplated activity of the\npolymerases under a few temperature and buffer conditions.\nAlthough the topic is interesting, the data resulting from the characterization is potentially useful, and the experimental\napproach is reasonable, I feel it is not sufficiently interesting and not sufficiently useful (no major insights, nor realistic\npotential applications), and the depth of analysis is insufficiently thorough to merit publication in Nature Communications.\nOne general concern with this type of experimental approach is that the data can be easily skewed by contamination. The\nauthors acknowledge likely contamination of the individual NTP mixes with other NTPs and it seems plausible that, in the\nabsence of a careful analytical characterization of the purity of the NTPs and other ingredients the results are significantly\naffected by contamination, skewed ratios of NTPs in the mixes, or even contamination with DNA. The authors state that the\npatterns were “robust and reproducible across replicates”, however the replicate date in the supplementary material show\nreadily noticeable differences. It is not clear how to interpret such differences, but in the absence of a well-defined metric, it\nseems that such a claim of reproducibility cannot be easily made.\nI do believe, a priori, that different polymerases and different environmental conditions can result in reasonably well defined\npatterns of nucleotide incorporation, but this is neither surprising nor necessarily as useful as the authors suggest. The\nauthors suggest or imply that it might be possible to control the “doodling” activity to the point of reaching something\napproaching sequence-controlled synthesis. In my opinion this is farfetched, particularly given the existence of polymerases\nlike TdT, which are more efficient for untemplated synthesis and are approaching full controllability. Even the title mentions\n“control of untemplated DNA polymerase activity” but this is far from what is actually shown.\nAlso missing from the manuscript is an explanation and detailed analysis/interpretation of the histograms obtained from the\nsequencing.", "reviewer_2": "The manuscript from Castle et al describes analysis of untemplated DNA synthesis by DNA polymerases. This is an\ninteresting but poorly understood area, and I think this study is very interesting and presents a new level of insight into the\nDNA made by this unusual mechanism. I think there a couple points that should be addressed and updated, but the paper is\nmostly suitable for publication and adds a significant contribution to the understanding of untemplated polymerase activity.\n1) The presence of G and C bases in the reactions containing only dA/TTP is puzzling, which the authors admit. The\nsuggestion of dNTP contamination seems pretty unlikely, as the G/C-containing sequences are pretty prevalent and a\nsignificant amount of the bases would have to be present. The reactions are heated at high temperature for very long times,\ncould some deamination of dA to dI give a signal read as G or C? Nucleotides are easily analyzed by mass spec and the\nvendor here likely could share any contaminant levels. I feel this is a mysterious result worth a bit more explanation.\n2) The authors acknowledge that the read length distributions might be biased to shorter products, but I think this is more\nsignificant than discussed. From the agarose gels in the paper it would seem the DNA products are overwhelmingly many\nkb in length, but the sequencing reads are predominantly very short. Could some of the longer DNAs be branched? Single-\nstranded? Would a HMW-focused library prep give reads more in line with the nature of the products? My concern is that the\nreads aren't reflective of the real nature of the DNA products and I think this could be examined more.\n3) The Family A and B polymerases used here give different transition frequencies, are these similar to published analysis of\nthe polymerases error spectra when fidelity and misincorporation are being examined?\n4) This is perhaps a bit pedantic, but I would suggest the authors not state they're using \"real-time PCR\" to measure the\nproduction of the DNA. They're doing isothermal reactions monitored by real-time fluorescence, no PCR is occurring at all\njust using a \"qPCR machine\" as a fluorimeter. I think these descriptions should be adjusted accordingly. Although it would\nbe interesting to see what DNA products would be produced if these reactions were thermocycled, the introduction of\ndenaturing steps might give reason to annealing of \"primer\" strands and different DNA."}, "author_response": "Response to reviewers (NCOMMS-24-60521)\nFind below our responses (in blue) to all the reviewers’ comments. We thank them for their valuable feedback\nand believe that our updates to the manuscript address all their concerns. We have also carefully reviewed\nthe entire manuscript and made minor changes throughout to improve readability. In the revised manuscript,\nsentences containing edits have been entirely highlighted in red to aid reviewing.\nReviewer #1:\nThis manuscript from Castle et al looks at the untemplated DNA synthesis activity of several DNA\npolymerases. In particular, they use long read synthesis to analyze the sequence composition resulting from\nthe untemplated activity of the polymerases under a few temperature and buffer conditions.\nAlthough the topic is interesting, the data resulting from the characterization is potentially useful, and the\nexperimental approach is reasonable, I feel it is not sufficiently interesting and not sufficiently useful (no major\ninsights, nor realistic potential applications), and the depth of analysis is insufficiently thorough to merit\npublication in Nature Communications.\nWe thank the reviewer for their consideration of our work. We were disappointed that unlike Reviewer\n2, they did not fully recognise the value of our study and the unique insights our experimental results\nprovide into the doodling process. However, we hope the substantial additional experiments within\nthe revision (including further nanopore sequencing and new atomic force microscopy) help them\nbetter appreciate our contribution to understanding this intriguing mechanism for DNA synthesis.\nOne general concern with this type of experimental approach is that the data can be easily skewed by\ncontamination. The authors acknowledge likely contamination of the individual NTP mixes with other NTPs\nand it seems plausible that, in the absence of a careful analytical characterization of the purity of the NTPs\nand other ingredients the results are significantly affected by contamination, skewed ratios of NTPs in the\nmixes, or even contamination with DNA. The authors state that the patterns were “robust and reproducible\nacross replicates”, however the replicate date in the supplementary material show readily noticeable\ndifferences. It is not clear how to interpret such differences, but in the absence of a well-defined metric, it\nseems that such a claim of reproducibility cannot be easily made.\nThe reviewer makes an important point that we agree requires further elaboration. To better\nunderstand the scale of contaminants in our reactions, we spoke with New England Biolabs who\nprovided all the key reagents used in our doodling reactions. Regarding contamination levels of the\ndNTPs, they confirmed that their individual dNTPs and dNTP mixes were ≥99% pure (determined by\nHPLC). It is therefore unlikely that this contamination could lead to significant amounts of doodled\nDNA containing these bases or skews in compositions. However, it could potentially account for the\nrecovery of sequences containing unexpected bases due to the sensitivity of the nanopore\nsequencing itself. This is further supported by the very small amounts of DNA produced by many of\nthese reactions (below the detection limit for the Qubit of 4 ng/µL), which compares to 100s–1,000s\nng/µL for many of the other doodling reactions. To highlight these points this section was updated:\n“It is unclear how DNA synthesis with bases not included in the reaction mix can take place, but could be\ndue to contaminants in the individual dNTP mixes or polymerase preparations used (the commercial provider\nof the reagents used here guarantee ≥99% pure dNTPs determined by HPLC) as well as the high sensitivity\nof the nanopore sequencing.”\nIn terms of the reproducibility of the experiments, we agree that the terminology “robust” may be\nmisinterpreted in relation to the experiments with the limited types of dNTPs. Our intention was to\nhighlight the consistent pattern in dominant A4T4 and A5T5 motifs produced. To ensure we are clear,\n1\nwe have reworded this section to highlight the consistent qualitative features, but increased\nquantitative variability in the composition of the doodled DNA pools:\n“In these cases, we found as expected that sequences were nearly entirely composed of these bases (87%\nof reads <250 nt and 99% of reads >250 nt). Interestingly, short and long reads were dominated by A4T4\nrepeats in addition to A5T5 repeats at a lower frequency (73% and 26%, respectively, across all reads >65 nt\nlong) (Figure 10c). In addition, while qualitative features of these repetitive sequence motifs were consistent\nacross experimental replicates (Supplementary Figure 13), much greater variation in the absolute factions of\nspecific motifs was seen compared to prior doodling reactions. Specifically, the A4T4 and A5T5 motifs that\ndominated both reactions saw drops of 16% and 10%, respectively across replicates. This suggests that\nthese reactions may be more sensitive to minor differences in the setup of the reactions and external\ncontaminants.”\nIn summary, while very low levels of contamination may be present in the dNTPs and enzymes, these\nare very unlikely to affect the strong and reproducible signals we see. We have updated the results\nto highlight these facts, as well as explain how the sensitivity of the nanopore sequencing enables\nvery weak signals, potentially produced by contaminants, to be observed when virtually no doodling\ntakes place.\nI do believe, a priori, that different polymerases and different environmental conditions can result in\nreasonably well defined patterns of nucleotide incorporation, but this is neither surprising nor necessarily as\nuseful as the authors suggest. The authors suggest or imply that it might be possible to control the “doodling”\nactivity to the point of reaching something approaching sequence-controlled synthesis. In my opinion this is\nfarfetched, particularly given the existence of polymerases like TdT, which are more efficient for untemplated\nsynthesis and are approaching full controllability. Even the title mentions “control of untemplated DNA\npolymerase activity” but this is far from what is actually shown.\nWe thank the reviewer for this useful comment and apologise if we were not clear in the way we\npresented our findings. Firstly, in response to the comment of well-defined patterns of nucleotide\nincorporation being neither surprising nor useful, we’d like to stress that until this study only a small\nnumber of individually cloned fragments of DNA from doodling reactions had been sequenced (see\nDOIs: 10.1093/nar/26.20.4652, 10.1007/s00792-014-0706-1, 10.1021/bi0489614). While biases in\nbiomolecular processes are common, precisely what these biases are is typically difficult or\nimpossible to predict a priori. Experiments like those presented in our paper are essential to uncover\nthis information and there can still be surprises in the specifics of the biases and the possible\nimplications these might have for the wider biology in which they are embedded. As highlighted by\nthe second reviewer’s comments, understanding these processes and providing the data to back up\npersonal beliefs or intuition is crucial in assessing whether such a phenomenon may have broader\nimpact or use.\nIn response to the second point on control and the emerging use of TdTs, we would like to stress\nthat we have been incredibly careful when presenting our results to not suggest that we envisage\nthis type of mechanism in the short-term being suitable for sequence perfect synthesis of long DNA\nfragments. Instead, as mentioned in the Discussion of the original submission, we envisage its value\nin guiding the synthesis of DNA pools with features in the compositional statistics of the sequences\npresent (see final paragraph of the Discussion). We made a concerted effort to use the term “guided”\nin key points and avoided any reference to terminology like “precise control”. We also disagree that\nit is inappropriate for the general term “control” to be used, given that the external environmental\nfactors we change do provide us with an ability to alter the outcomes in a reproducible way.\nAs for the use of doodling for DNA synthesis as being “farfetched” and TdTs being a better approach,\nwe do see some specific use-cases that were likely not sufficiently highlighted or elaborated on in\nthe initial submission. Specifically, the ability for doodling DNAPs to produce highly repetitive\nsequences. Repeats are very difficult to synthesize using current approaches (including TdTs), but\n2\nare important structurally in virus genomes (e.g., in terminal repeats) and are present throughout the\nhuman genome (e.g., Alu repeats are 11% of the human genome and modify gene expression). Being\nable to synthesize such sequences would fill a gap in current TdT capabilities and could help us to\nfurther understand the effects of these repeats in nature and engineered biological systems.\nFurthermore, the ability for DNAPs to perform both untemplated and templated synthesis offers yet\nfurther interesting possibilities that TdT technologies cannot support (e.g., de novo synthesis and\namplification in a single reaction). These capabilities in our mind make it foolish to discount DNAP-\nbased enzymatic synthesis as a dead-end that is not worth pursuing.\nTo try and address the reviewer’s concern, we have expanded the Discussion to be more explicit\nabout the value we see in this phenomenon for DNA synthesis and carefully reviewed the entire\nmanuscript to ensure our use of the word “control” is warranted:\n“The vertebrate immune system uses TdT to add nucleotides without any template in order to increase\nantigen receptor diversity 35 and was previously considered the only enzyme able to generate DNA without a\ntemplate. Enzymatic DNA synthesis using TdT has spawned several successful companies such as\nMolecular Assemblies, DNA Script, and Ansa Biotechnologies 36 who have developed specialized protocols,\n3’-protection groups 37–39, and re-engineered TdT with more rapid nucleotide addition (Patent\nWO/2019/135007), modified catalytic activity 40, and with dNTPs tethered to the enzyme 28. These advances\nhelp to mitigate some of the limitations of TdTs, including the bias towards the addition of particular\nnucleotides 41. Development of DNAP-based enzymatic DNA synthesis could similarly improve the control of\nuntemplated DNA synthesis and provide further benefits. For example, TdT approaches typically struggle to\nproduce highly repetitive and structurally prone sequences that are known to be important structurally in virus\ngenomes (e.g., in terminal repeats) and which are present throughout the human genome (e.g., Alu repeats\nare 11% of the human genome and modify gene expression). The demonstrated ability for doodling by\nDNAPs to effectively synthesize repetitive sequences would fills a gap in current TdT capabilities and could\nhelp us to further understand the effects of repeat sequences in nature and engineered biological systems.\nBeyond sequence complexity, the yield, purity and length of DNA synthesized by any methodology\ndepends on the efficiency of each cycle of nucleotide addition. Despite the excellent reported efficiency from\nDNA Script of 99.7% 42, mathematically, the yield of products of 1 kb in length is only ~5%, and even 99.9%\nefficiency yields <37% of 1 kb length products 36, not taking into consideration the error rate. Longer DNA\nconstructs have been made using DNA assembly methods, namely a 10 kb construct from Ribbon Biolabs\nusing Gibson assembly (Patent WO/2019/073072) and whole genomes using yeast-based DNA assembly\n43,44. However, assembly of DNA fragments can be laborious and expensive so the development of novel\ntechnologies to write longer and more accurate DNA constructs will be vital in advancing synthetic genomics.\nFurthermore, while the fidelity of doodling at present means it is impossible to synthesise long and defined\nsequences, the ability of DNAP-based methods to both carry out templated and untemplated DNA synthesis,\ncould open up new avenues for DNA production that exploit de novo synthesis and amplification concurrently\n– features that no existing technologies are able to combine.”\nAlso missing from the manuscript is an explanation and detailed analysis/interpretation of the histograms\nobtained from the sequencing.\nWe apologise to the reviewer for having not provided sufficient analysis/interpretation of the read\nlength histograms obtained from the sequencing. We had initially focused most of our efforts on\nunderstanding the patterns in the sequences produced but have attempted to address this concern\nin the revision by expanding the Results when these are introduced to cover their shapes in more\ndepth. Given that the size distributions capture statistics related to the underlying generative\nprocess, we have also made comments relating to the likely rates of synthesis and possible inhibitory\neffects seen for very long fragments.\nRelated to this point, we also carried out further experiments to try and understand the discrepancy\nbetween the gel images that showed large quantities of very long DNA fragments (10,000s–100,000s\nnt) to the read length distributions from the nanopore sequencing. To do this, we performed\n3\nadditional atomic force microscopy (AFM) experiments on the doodled DNA pools to better\nunderstand the physical structure of the molecules produced. Interestingly, we discovered highly\nbranched, tree-like structures that could account for the observed discrepancies. Specifically, the\nlarge smears seen in the gels that typically would be indicative of wide-ranging lengths of DNA\nproduct are likely an artifact of the physical structures within the DNA pools themselves. Moreover,\nthe close correspondence in the median molecule/read lengths from the AFM images and nanopore\nsequencing (518 nt versus 552 nt, respectively), suggests that the read length distributions are\ncapturing a realistic picture of the true lengths of the doodled DNA molecules. This new experimental\ndata is presented and analysed as a new section of the Results called “Physical structure of doodled\nDNA pools”.\nReviewer #2:\nThe manuscript from Castle et al describes analysis of untemplated DNA synthesis by DNA polymerases.\nThis is an interesting but poorly understood area, and I think this study is very interesting and presents a new\nlevel of insight into the DNA made by this unusual mechanism. I think there a couple points that should be\naddressed and updated, but the paper is mostly suitable for publication and adds a significant contribution to\nthe understanding of untemplated polymerase activity.\nWe are grateful to the reviewer for their careful assessment of our work and are pleased that they\nrecognised its valuable contribution, providing the most in-depth picture of DNAP doodling activity\nto date. Below we have outlined how the minor comments raised have been fully addressed in our\nrevision and some of the additional experiments performed. This includes new nanopore sequencing\nand atomic force microscopy that provides to our knowledge the first “look” at the structure of the\ndoodled DNA products we synthesise.\n1) The presence of G and C bases in the reactions containing only dA/TTP is puzzling, which the authors\nadmit. The suggestion of dNTP contamination seems pretty unlikely, as the G/C-containing sequences are\npretty prevalent and a significant amount of the bases would have to be present. The reactions are heated at\nhigh temperature for very long times, could some deamination of dA to dI give a signal read as G or C?\nNucleotides are easily analyzed by mass spec and the vendor here likely could share any contaminant levels.\nI feel this is a mysterious result worth a bit more explanation.\nThe reviewer makes an excellent point about the high temperatures that the reactions are run at and\nthe possibility of deamination of deoxyadenosine (dA) to deoxyinosine (dI) which could influence the\nnanopore sequencing. To assess whether dI nucleotides would be resolved as G or C bases, we\ncarried out a new experiment with pairs of complementary synthesised primers 43 nt in length that\ncontained a run of three dI nucleotides in their centre on one of the strands. Pools of these oligos\nwere annealed and nanopore sequenced. Analysis of the sequencing data showed that the flanking\n20 nt were accurately resolved, while the three dI nucleotides were typically basecalled as C or G\nbases with the most common resolved sequences being ‘CGC’ that was seen for 32% of all reads for\nthat strand. This contrasted with the opposite strand where most reads (72%) were correctly called\nas ‘TTT’ although a small fraction (11%) were called as ‘CCC’. This miscalling is likely due to the effect\nthat the complementary strand containing the modified nucleotides has during sequencing.\nAs recommended by the reviewer, we also spoke with New England Biolabs about contamination\nlevels in the dNTPs used for our experiments. They confirmed <1% contamination (measured by\nHPLC), which while low, would be measurable with the highly sensitive nanopore sequencing carried\nout. Even so, it is unlikely this would lead to significant amounts of doodled DNA containing these\nbases. Furthermore, we carried out a statistical analysis of the E. coli BL21(DE3) genome to assess\nwhether, contamination by cells used in the production of the polymerases could act as a source of\nthe repetitive motifs we recovered. We found that only a single motif was found once within the\ngenome meaning it cannot be the source of the diverse repetitive sequences we see.\n4\n2) The authors acknowledge that the read length distributions might be biased to shorter products, but I think\nthis is more significant than discussed. From the agarose gels in the paper it would seem the DNA products\nare overwhelmingly many kb in length, but the sequencing reads are predominantly very short. Could some\nof the longer DNAs be branched? Single-stranded? Would a HMW-focused library prep give reads more in\nline with the nature of the products? My concern is that the reads aren't reflective of the real nature of the\nDNA products and I think this could be examined more.\nThe reviewer again makes some excellent suggestions about further analysis that could be carried\nout to better understand the nature of the doodled DNA. To directly address these, we performed two\nsets of additional experiments making use of (i) atomic force microscopy (AFM) and (ii) nanopore\nsequencing using a high-molecular weight (HMW) preparation protocol.\nResults from the AFM highlighted a fascinating insight into the structure of the doodled DNA,\nshowing a highly branched structure that is likely formed through base pairing of repetitive sequence\nmotifs across different doodled DNA molecules. This leads to tree-like structures that would help\nexplain the large smears seen in the agarose gels because the tree-/mesh-like structures would run\nsignificantly slower and give the false impression of very large molecules. We do still see some very\nlong products (~2800 nt) in the AFM data. However, the majority are much shorter (<2,000 nt), which\nis more in line with the distributions we see from the nanopore sequencing. We added a new section\nto the Results called “Physical structure of doodled DNA pools” to describe this new AFM data and the\nfascinating structural insights it provides. To our knowledge, this represents the first time that AFM\nhas been performed on doodled DNA and the first comprehensive images and analysis of the\nbranching structures produced, adding further to the novelty and importance of this work.\nWe also carried out nanopore sequencing experiments that make use of approaches to help ensure\nthe stability of HMW products during the sequencing. These experiments, produced DNA fragment\nlength distributions with a slightly larger median value, further supporting the idea that the very long\nDNA products seen in the agarose gels are in fact likely DNA nanostructures formed between shorter\ndoodled DNA fragments. This analysis was added as a new Results section called “Assessing the\ncapture of high molecular weight DNA”.\n3) The Family A and B polymerases used here give different transition frequencies, are these similar to\npublished analysis of the polymerases error spectra when fidelity and misincorporation are being examined?\nThis is a very interesting question, given that our data spans both Family A (Taq) and B (Vent,\nTherminator) polymerases. To assess possible biases, we calculated average base transition\nprobabilities to complement the molecule-level transition probabilities shown in the blue panels of\nFigures 2 and 4 of the original manuscript. In comparison to the known error spectra of these\npolymerases, we found clear differences. This suggests that the structural constraints for doodling\nactivity are very different to those affecting DNA copying fidelity and that these biases can be shaped\nthrough DNAP engineering. A new section call “Analysis of DNA polymerase transition frequencies” has\nbeen added to present this new data and analysis.\n4) This is perhaps a bit pedantic, but I would suggest the authors not state they're using \"real-time PCR\"\nto measure the production of the DNA. They're doing isothermal reactions monitored by real-time fluorescence,\nno PCR is occurring at all just using a \"qPCR machine\" as a fluorimeter. I think these descriptions should be\nadjusted accordingly. Although it would be interesting to see what DNA products would be produced if these\nreactions were thermocycled, the introduction of denaturing steps might give reason to annealing of \"primer\"\nstrands and different DNA.\nWe thank the reviewer for this suggestion and agree that “monitoring of real-time fluorescence” is a\nmore accurate description of our methodology. We have made appropriate changes throughout the\n5\nmanuscript and in the figures to clarify this point and be more explicit about how qPCR machines\nwere used.\nIn terms of adding thermocycling to the reaction, we decided to carry this out for both Taq and Vent,\nalternating between 65°C for 20 mins and 74°C for 20 min for 9 cycles. We expected this to accelerate\nthe production of the repetitive sequence motifs, but instead it resulted in a complete breakdown in\ntheir formation for both polymerases. Overall, virtually no doodling was seen with an order of\nmagnitude less DNA produced, with most of it being mixtures of genomic contaminants from the E.\ncoli genome (likely from the commercially provided enzymes we were using). We have added a new\nsection to the Results called “Impact of cycling thermal conditions” to discuss these new experiments\nand analysis.\n6"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has been significantly extended with the addition of new experimental results as well\nas extensive revisions to the text, in both cases to address concerns from the reviewers. In my opinion, the\nrevisions have significantly improved the manuscript, particularly by addressing the possibility that some of\nthe experimental results may be due to DNA contamination. Given these improvements, I recommend that\nthe manuscript be published in Nature Communications with minor changes. In particular:\n1.In my view, the value of the “doodling” to DNA synthesis is oversold. For example, the statement (line 496)\nto the effect that it could be disruptive to current chemical or TdT-based synthesis cannot be justified by the\nresults presented. I suggest that this type of statement, here and elsewhere, be toned down a couple of\nnotches, as there is almost no conceivable path that would allow doodling to reach even the level of control\navailable via current TdT variants or chemical synthesis.\n2.I think the interest in the author's results will be more in the direction of potential signatures of doodling in\nnatural systems. However, even here, I think the authors need to be more careful with unsupported\nstatements. The statement (lines 530 to 539) regarding hits in nucleotide databases of e.g. CTATAG,\nneeds to be either removed or supported with a statistical analysis. Any 6mer is going to be present in very\nlarge numbers in any large genome, and even if the doodling mechanism had some role in early evolution,\nit seems likely that a few billion years of evolution would have any evidence of this.", "reviewer_2": "The authors have, in my view, done an excellent job responding to reviewer comments and have added a\nsignificant amount of new data. These additions result in a more complete and clearer investigation of the\ninteresting activities of the DNA polymerases presented. The AFM data in particular helps clear up one of the\nbiggest questions I had from the first version. So, these branches must not be 5' extensions but rather something else? More a note of curiosity than something the authors need to address.\nOverall, I think this is now an impressive collection of data that explores the findings very thoroughly and with\nmore than enough discussion of the various hypotheses and potential applications of the phenomenon. In\nmy opinion this manuscript is suitable for publication as is and serves as an important contribution to this\nfield."}, "author_response": "Response to reviewers (NCOMMS-24-60521A)\nFind below our responses (in blue) to the final minor comments from the reviewers. We thank them for their\ncontinued and valuable feedback throughout the review process. We have also carefully reviewed the entire\nmanuscript and made minor changes throughout to address additional editorial requests to ensure the format\nis appropriate for publication.\nReviewer #1:\nThe revised manuscript has been significantly extended with the addition of new experimental results as well\nas extensive revisions to the text, in both cases to address concerns from the reviewers. In my opinion, the\nrevisions have significantly improved the manuscript, particularly by addressing the possibility that some of\nthe experimental results may be due to DNA contamination. Given these improvements, I recommend that\nthe manuscript be published in Nature Communications with minor changes. In particular:\nWe thank the reviewer for their further consideration of our work and their appreciation of our efforts\nto expand the scope of the work to address their previous concerns.\n1. In my view, the value of the “doodling” to DNA synthesis is oversold. For example, the statement (line 496)\nto the effect that it could be disruptive to current chemical or TdT-based synthesis cannot be justified by the\nresults presented. I suggest that this type of statement, here and elsewhere, be toned down a couple of\nnotches, as there is almost no conceivable path that would allow doodling to reach even the level of control\navailable via current TdT variants or chemical synthesis.\nWe agree that doodling, as presented, is unlikely to reach the accuracy seen in current TdT\ntechnologies, even the medium term. To ensure we do not overstate its possibilities, as suggested\nby the reviewer, we have toned down statements related to future sequence perfect synthesis of DNA\nby doodling throughout the manuscript.\n2. I think the interest in the author's results will be more in the direction of potential signatures of doodling in\nnatural systems. However, even here, I think the authors need to be more careful with unsupported\nstatements. The statement (lines 530 to 539) regarding hits in nucleotide databases of e.g. CTATAG, needs\nto be either removed or supported with a statistical analysis. Any 6mer is going to be present in very large\nnumbers in any large genome, and even if the doodling mechanism had some role in early evolution, it seems\nlikely that a few billion years of evolution would have any evidence of this.\nWe agree that in the short term, the impact of doodling on genome content is an interesting and\naccessible direction to explore and that careful consideration of confounding factors will be\nessential. We have expanded the Discussion a little to cover these important points: “An intriguing\nfuture direction for this work would be to perform a detailed analysis of common doodled motifs throughout\nknown genome sequence space, to assess whether doodling might have played a role in fuelling early\nevolution (e.g., through comparisons of motifs in genes of early origins) acting as a feedstock for natural\nselection to act upon. Any such analysis would need to employ careful statistical analyses to ensure other\nevolutionary processes did not confound the results. It may also be more productive to consider using bottom-\nup synthetic biology approaches in which systems can be more thoroughly observed and precisely controlled\nover time.”\nIn relation to the comment on the analysis we did of the repetitive sequence motifs, we did not search\nfor a 6-mer sequence across the NCBI, but 5 copies of the 6-mer sequences (a 30-mer sequence).\nThis is very unlikely to occur by random chance (p = 8.67 × 10–19). Due to this confusion, we have\nelaborated on this sentence to highlight our point more clearly: “A quick BLAST search for the 30-mer\nsequence (CTATAG)5, a long repeat of the motifs we find, across the NCBI core nucleotide database yields\nlarge numbers of hits from across the tree of life, with many being perfect matches. Such sequences are\n1\nunlikely to occur by random chance (p = 8.67 × 10–19, assuming all nucleotides are equally probable).\nHowever, it remains to be seen if these patterns are the result of prior doodling activity or if they arose through\nother means.”\nReviewer #2:\nThe authors have, in my view, done an excellent job responding to reviewer comments and have added a\nsignificant amount of new data. These additions result in a more complete and clearer investigation of the\ninteresting activities of the DNA polymerases presented. The AFM data in particular helps clear up one of the\nbiggest questions I had from the first version. The comment about the branching structure of the DNA\nobserved by AFM is an interesting on. Our hypothesis is that these are not truly branched molecules,\nbut instead, the branches are in fact caused through base pairing of two separate ssDNA molecules\nat sequences containing the repetitive motifs we observe in the sequencing. This is supported by\nsome of the raised heights of the molecules at these points in some of the AFM images and is\nmentioned in the main text: “Closer inspection of some of these imaged molecules showed increased\npeaks in height at points where branches occurred (Figure 5b, white arrows). This suggests that these regions\nmay be double stranded, supporting the idea of base pairing between separate ssDNA molecules, or through\nthe formation of hairpins within a single ssDNA molecule.”\n2"}]

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