Supplementary Information of Experiment

1. Experimental work content

The concentration of each DNA was measured by absorbance and diluted with UPW before use.
All components were mixed in a PCR tube to a total volume of 20 µL.
DNA oscillation was confirmed by real-time PCR (~12–24 h, 46 °C) and quantified by fluorescence.
EvaGreen: fluorescence increases with dsDNA; used to quantify duplex P (present in the 4× buffer).
DY530: a fluor attached to the 3′ end of normal G; N–G binding reduces fluorescence; not attached to amino-acid–modified G.

2. Details of Buffer Composition

In the actual experiments, this 4× buffer was diluted to 1× by mixing it with other components (DNA, enzymes, etc.) to achieve the final reaction volume.
The final concentrations of each component in the 1× buffer are also listed in the table below.

Component	Stock conc.	Add (µL)	Final in 4×	Final in 1×
Tris-HCl (pH 8.8)	1000 mM	40	80 mM	20 mM
(NH₄)₂SO₄	1000 mM	20	40 mM	10 mM
KCl	1000 mM	20	40 mM	10 mM
NaCl	1000 mM	100	200 mM	50 mM
MgSO₄	1000 mM	16	32 mM	8 mM
dNTPs	10 mM	80	1.6 mM	0.4 mM
Synperonic	10 %	20	0.4 %	0.1 %
Netrospin	2	2	0.008	0.002
EvaGreen	20×	100	4×	1×

3. Details of the DNA oscillation verification experiment

3.1 Experiment 3-1

Objective of the experiment;

The oscillations were investigated using the composition parameters described in the previous study [1].

Composition table

Graph of experimental results

Horizontal axis: Time; Vertical axis: Fluorescence intensity.
The graph on the left represents P, while the graph on the right represents N.
Oscillatory behavior was examined under conditions where the concentration of the template DNA, G, was set to 60 nM, 110 nM, 160 nM, and 210 nM, respectively.
To evaluate the enzymatic activity of ttRecJ, an independent experiment was performed using Reaction 3 alone.
Oscillatory behavior was observed immediately after the reaction started; however, the concentration of P subsequently increased. In other words, P did not decrease, while the proportion of N declined.

3.2 Experiment 3-2

Objective of the experiment

Since oscillations were not observed in the previous experiment (Experiment 3-1), it was assumed that the concentrations of N and P were not appropriate. Therefore, the concentrations of these two DNAs were varied to examine whether oscillatory behavior could be observed.

Composition table

Graph of experimental results

The concentration of G was fixed at 160 nM across all reactions (R1–R4).
R1: N 50 nM, P 150 nM
R2: N 75 nM, P 225 nM
R3: N 100 nM, P 300 nM
R4: N 125 nM, P 375 nM
A sharp increase in the proportion of P was observed around 6 hours after the start of the reaction (as shown in the left graph).

3.3 Experiment 3-3

Objective of the experiment

Based on the results of Experiment 3-2, it was assumed that the ratio of N to P (1:3) provided an insufficient amount of N. Therefore, the ratio was adjusted to examine the effect of this change on the oscillatory behavior.

Composition table

Graph of experimental resultsThe concentrations of N and P in each reaction system were maintained within the range of 100–200 nM. The concentration of G was fixed at 160 nM, the same as in the previous experiment.

Even when the ratio of N to P was varied, the abrupt increase in the proportion of P persisted, and no oscillatory behavior was observed.

3.4 Experiment 3-4

Objective of the experiment

Due to the strong effect of Polymerase (Bst Large Fragment), degradation by Exonuclease (ttRecJ) is insufficient, resulting in a pronounced increase in P and the absence of oscillatory behavior.
It is anticipated that increasing the proportion of ttRecJ could produce behavior more closely resembling oscillations.

Composition table and Graph of experimental results

Description of the results
The DNA concentration was verified by measuring absorbance during dilution.
Compared with previous results, no significant changes were observed, and oscillatory behavior was not detected.

3.5 Experiment 3-5

Objective of the experiment

Oscillatory behavior was examined using ttRecJ at different concentrations, as well as with another ttRecJ preparation previously available in the laboratory.
The ttRecJ variants were as follows:
2.5 μM dilution (RJ1)
Stock solution (8.7 μM) (RJ2)
Laboratory-available ttRecJ (RJ3)

Composition table and Graph of experimental results

Description of the results
Oscillations were observed with RJ3 during the first 6 hours.

3.6 Experiment 2-6

Objective of the experiment

Under the previously used compositional conditions, the action of Polymerase is dominant, and degradation by ttRecJ is insufficient, which is assumed to prevent oscillations.
Since mild oscillations were observed with RJ3, the type and concentration of ttRecJ were fixed, and the reaction was examined while varying the concentration of Polymerase.

Composition table and Graph of experimental results

Description of the results
Oscillatory waveforms were observed throughout the entire measurement period at 0.5% Polymerase.

3.7 Experiment 3-7

Objective of the experiment

The reaction system was examined by varying the concentration of Polymerase from 0.1% to 0.75%, compared with 1%.

Composition table and Graph of experimental results

Description of the results
Oscillations were observed at 0.75%, 0.5%, and 0.3% Polymerase.
The waveform at 0.75% Polymerase most closely matched the oscillation period reported in the reference [1].
Furthermore, oscillations at 0.75% exhibited less damping compared with those at other concentrations.

4. DNA oscillations with amino acid–modified template DNA

4.1 Experiment 4-1

Objective of the experiment

Oscillations were examined using unmodified G, G modified with an amino group, and G conjugated with Boc-protected Lysine, in order to assess whether these modifications affect the oscillation period.

Composition table and Graph of experimental results

Description of the results
Polymerase was used at 0.75%, and ttRecJ was RJ3.
A shift in oscillation period was observed for each modified G compared with unmodified G.
Compared with previous experimental results, the oscillation period of G was different.

4.2 Experiment 4-2

Objective of the experiment

Description of the results
Experiments were conducted under the same compositional conditions as Experiment 3-2, extending the PCR measurement time to 24 hours in order to observe the oscillation period over a longer duration.
The same experiments were also performed using RJ1 in place of RJ3.

Composition table and Graph of experimental results

Description of the results
The 4× buffer prepared on September 4 was freshly remade.
It was suspected that the freshly prepared buffer on the day of the experiment was responsible.
Specifically, when taking dNTPs from the stock to prepare the buffer, they may not have been mixed thoroughly.

4.3 Experiment 4-3

Objective of the experiment

The buffer was carefully remade, and the experiment was conducted under the same conditions as Experiment 4-2.

Composition table

The composition was the same as that used in Experiment 4-2.

Graph of experimental results

Description of the results
Under the RJ1 condition, an increase in P was observed.
Since Polymerase is dominant, the rate of DNA degradation by RJ1 is likely lower than the rate of P accumulation.
Under the RJ3 condition, the first hour showed behavior similar to G–Lysine, but the proportion of P increased thereafter. Compared with the results on Experiment 4-1, the oscillation period of G under the RJ3 condition was delayed.

4.4 Experiment 4-4

Objective of the experiment

The oscillation period of DNA oscillations was examined using G conjugated with Glycine (G–Glycine), in which the amino group of G is linked to Glycine.

Composition table and Graph of experimental results

Description of the results
DNA oscillations were not observed with the template DNA conjugated with Glycine.
Regarding the pulse observed with G, the timing was delayed compared with previous results.

5. Expression and purification of ttRecJ

Express ttRecJ in E. coli Rosetta (DE3)pLysS grown in LB + ampicillin (100 μg/mL) at 37 °C to OD₆₀₀ ≈ 0.4, then induce with IPTG (final 0.4 mM) and shake at 30 °C for ~3 h.
Harvest at 5,000 × g for 15–20 min (4 °C); wash once with Buffer A and re-spin. Keep pellet on ice (or store at −80 °C).
Resuspend in Buffer A (e.g., 10 mL per 500 mL culture) and lyse on ice by sonication. Clarify at 5,000 × g for 30 min (4 °C) and 0.22 μm-filter the supernatant.
Ni–NTA binding: Incubate the supernatant with pre-equilibrated Ni–NTA resin (≈500 μL, 50% slurry) for 1 h at 4 °C with gentle rotation; spin 600 × g for 4 min and discard flow-through.
Wash: Wash 2–3× with Buffer A + 20 mM imidazole (~5 mL each); gentle inversion 10–15 min at 4 °C, then spin and discard.
Elution: Elute with Buffer A + 200 mM imidazole in ~200 μL fractions; main protein typically appears in fractions #2–#3 (verify by SDS–PAGE/A₂₈₀).
Buffer exchange & storage: Desalt into storage buffer (10 mM potassium phosphate pH 6.8, 0.1 mM EDTA, 10% glycerol), aliquot, keep 4 °C (short-term) or snap-freeze and store at −80 °C.
Working dilution buffer (example, 300 μL): 10 mM Tris–HCl pH 7.4, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol; fill with UPW to volume.
Notes: Keep everything cold; minimize freeze–thaw cycles; use low-bind tubes; adjust imidazole in wash/elution as needed for purity/yield.

Working dilution buffer composition (total 300 μL)
Component	Stock	Volume (μL)	Final
Tris–HCl (pH 7.4)	1 M	3	10 mM
KCl	1 M	30	100 mM
EDTA	10 mM	3	0.1 mM
DTT	100 mM	3	1 mM
Triton X-100	10%	3	0.1%
Glycerol	—	150	50% (v/v)
Ultra-pure water (UPW)	—	108	—
Total	—	300	—

6. Reference

Fujii T, Rondelez Y. Predator–Prey Molecular Ecosystems. ACS Nano (2013).

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