Research ArticleChemistry

Programmable dynamic steady states in ATP-driven nonequilibrium DNA systems

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Science Advances  19 Jul 2019:
Vol. 5, no. 7, eaaw0590
DOI: 10.1126/sciadv.aaw0590
  • Fig. 1 ATP-fueled dynamization of phosphodiester bonds by simultaneous action of two antagonistic DNA enzymes for transient dynamic covalent polymerization of DNA strands with tunable lifetimes and adjustable steady-state dynamics.

    (A) Short telechelic DNA monomers, M1, with 4-nt self-complementary ssDNA ends are covalently joined via T4 DNA ligase–catalyzed phosphodiester bond formation under consumption of two ATP fuel molecules. This ligation forms the recognition site (highlighted by the orange box) of the endonuclease BamHI, which counteracts ligation by catalyzing the cleavage (restriction path as red line) of the just formed phosphodiester bonds. Simultaneous ligation and cutting at this site creates a dynamic covalent bond until the ATP runs out. (B) Transient growth of dynamically polymerizing DNA chains in a closed system is achieved by a faster ligation than restriction reaction (vactvdeact). The lifetime is coupled to the ATP fuel and can be tuned together with the DySS properties of the dynamic covalent DNA chains under biocatalytic control.

  • Fig. 2 ATP-fueled transient DySS polymerization of dynamic covalent DNA chains with a tunable lifetime.

    (A) Time-dependent GE shows transient lifetimes programmed by ATP fuel concentration (0.1 to 1.0 mM ATP, left to right). Lane assignment: 1, 50-bp ladder; 2, 1-kbp ladder; 3, 0 min; 4, 10 min; 5, 1 hour; 6, 9 hours; 7, 24 hours; 8 to 16, 2 to 10 days (daily interval). (B) Gray scale profiles extracted from GE at 0.4 mM ATP (panel ii) quantify the transient, reversible shifts of molecular weight (top, growth; bottom, decline), which is used to calculate the mass-weighted average chain length (bp¯w) for each kinetic aliquot. (C) The development of bp¯w over time reveals increasing lifetimes of the transient DNA polymerization with a constant steady-state chain length of around 1000 bp under the given enzymatic conditions when increasing the ATP concentration from 0.1 to 1.0 mM. Lines are guides to the eye. (D) The lifetime scales linearly with the amount of supplied ATP. Error bars result from averaging triplicate measurements. Conditions: 0.05 mM M1, 41.25 WU of T4 DNA ligase, 900 U of BamHI, and varying amounts of ATP at 25°C in the enzyme reaction buffer.

  • Fig. 3 Programming the transient ATP-fueled DySS polymerization of DNA chains by changing the dynamics of ligation and cleavage under biocatalytic and thermal control.

    The starting configuration of the systems comprises 0.05 mM M1 (38 bp), 41.25 WU of T4 DNA ligase, 900 U of BamHI, and 0.1 mM ATP in the enzyme reaction buffer at 16°, 25°, and 37°C. Each of these parameters is systematically varied to tune the dynamics of the transiently evolving chains: Increase of (A) T4 DNA ligase, (B) BamHI, or (C) of both enzymes symmetrically shifts the kinetic balance of the competing reactions either to the ligation or restriction side, leading to different DySSs and lifetimes (0.1 mM ATP, 25°C). (D) Dynamics and ATP-dependent lifetimes can be further controlled by temperature: top, 16°C, dynamics slow down; bottom, 37°C, dynamics speed up. (E) Comparison of the temperature-dependent temporal development of the average chain length bp¯w for selected ATP concentrations: top, 1.0 mM; bottom, 0.1 mM. (F) Time-dependent GE showing reactivation of transient chain growth by addition of ATP (both cycles fueled with 0.1 mM ATP, 37°C). (G) The corresponding plots of bp¯w over time demonstrate identical dynamic system behavior for the second cycle. Control experiments elucidate ATP as the driving force for successful reinitiation. Lines in all graphs are drawn as a guide to the eye.

  • Fig. 4 Adaptive DySSs and molecular exchange in the dynamic covalent DNA bond system.

    (A to C) Intermolecular exchange between two different dimer duplexes DS (72 bp) and DL (100 bp) upon enzymatic dynamization of the dynamic covalent restriction site. (A) DNA species formed during the transient ATP-fueled dynamization. (B) GE of ATP-fueled dimer exchange kinetics (37°C) shows the transient occurrence of a hybrid species D* (84 bp) and provides evidence for molecular reshuffling of the fragments. (C) Gray scale profiles highlight D* in green. Conditions: 0.5 μM DL, 0.5 μM DS, 37°C. (D to F) Dynamic sequence shuffling between fluorescently labeled DNA chains proves intermolecular subunit exchange also on the polymer level. (D) Two homopolymers, short fluorescein-tagged PFl (green) and long Cy5-tagged PCy5 (red), were mixed together and turned into a random copolymer upon DySS activation. (E) The shuffling process and evolution into a DySS polymer is followed by selective multicolor GE. The multicolor GE is a composite image of the fluorescein (green) and the Cy5 (red) channel. The fluorescent oligomers show different migration distances and can be distinguished from each other (compare first two lanes of pure homopolymers PFl and PCy5). A randomized DySS sequence appears in orange color and by homogenization of the band migration. (F) Gray scale analysis of the individual fluorophore channels and the composite reveals the different compositions of the static (0 hour) and dynamic (48 hours) polymer “mix” (framed sections in GE). Convergence of the initially separated bands into one DySS band, for instance, the heptamer fraction (no. 7), demonstrates successful sequence shuffling and subunit exchange. Conditions: 5.0 μM MFl in PFl, 2.5 μM MCy5 in PCy5, 37°C. (G to I) Adaptive DySSs monitored by FRET duplex activation (fig. S9 and S10 for details). (G) The dynamic covalent bond was equipped with the Cy3/Cy5 FRET pair to report the DySS ligation level via the FRET ratio Imax,Cy5(acceptor)/Imax,Cy3(donor). The FRET ratio can be translated into a fraction of ligation, which is effectively an ensemble average steady-state bond strength. (H) Formation of different DySSs in dependence of the enzyme ratio [T4]/[BamHI] at 25°C: variation of the T4 DNA ligase (left, [BamHI] = 100, U = constant) and BamHI (right, [T4] = 2.29, WU = const). (I) In situ adaptation of the DySS in a transient ATP-fueled FRET duplex activation by sequential addition of individual enzymes. Conditions: 1 μM FCy3, 1 μM FCy5, 25°C, λexc = 505 nm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw0590/DC1

    Supplemental Materials and Methods

    Experimental Protocols

    Supplementary Note A. Development of the conditions for the dynamic reaction network by characterization of the individual enzyme reactions

    Supplementary Note B. Routine of GE analysis: From the agarose gel to an average chain length

    Supplementary Note C. ATP-fueled transient, dynamic steady-state DNA polymerization system

    Supplementary Note D. DySS and molecular exchange in ATP-fueled dissociative dynamic covalent DNA systems

    Table S1. Oligonucleotide sequences.

    Fig. S1. Hybridization of the self-complementary ends of the DNA monomer strands M1 in dependence of temperature and ligation reaction catalyzed by T4 DNA ligase.

    Fig. S2. Ligation kinetics of the DNA chain growth as a function of T4 DNA ligase concentration.

    Fig. S3. Ligation kinetics of the DNA chain growth as a function of ATP concentration.

    Fig. S4. Time-dependent T4 DNA ligase catalyzed ligation reaction.

    Fig. S5. Restriction kinetics of the DNA chain cleavage as a function of BamHI concentration.

    Fig. S6. Routine for analysis of GE data: From the agarose GE to an average DNA chain length bp¯w.

    Fig. S7. Control of dispersity in the DySS DNA polymerization system.

    Fig. S8. Refueling experiments of the transient DySS DNA polymerization system.

    Fig. S9. Average chain length in the transient DySS DNA polymerization system in dependence of the concentration of the DNA monomer M1.

    Fig. S10. Characterization of the FRET duplex F and its cleaved and religated DNA fragments as used for in situ modulation of the DySS.

    Fig. S11. ATP-dependent temporal control of the dynamic DNA bond with transient DySS FRET duplex formation.

    References (40, 41)

  • Supplementary Materials

    This PDF file includes:

    • Supplemental Materials and Methods
    • Experimental Protocols
    • Supplementary Note A. Development of the conditions for the dynamic reaction network by characterization of the individual enzyme reactions
    • Supplementary Note B. Routine of GE analysis: From the agarose gel to an average chain length
    • Supplementary Note C. ATP-fueled transient, dynamic steady-state DNA polymerization system
    • Supplementary Note D. DySS and molecular exchange in ATP-fueled dissociative dynamic covalent DNA systems
    • Table S1. Oligonucleotide sequences.
    • Fig. S1. Hybridization of the self-complementary ends of the DNA monomer strands M1 in dependence of temperature and ligation reaction catalyzed by T4 DNA ligase.
    • Fig. S2. Ligation kinetics of the DNA chain growth as a function of T4 DNA ligase concentration.
    • Fig. S3. Ligation kinetics of the DNA chain growth as a function of ATP concentration.
    • Fig. S4. Time-dependent T4 DNA ligase catalyzed ligation reaction.
    • Fig. S5. Restriction kinetics of the DNA chain cleavage as a function of BamHI concentration.
    • Fig. S6. Routine for analysis of GE data: From the agarose GE to an average DNA chain length bp¯w.
    • Fig. S7. Control of dispersity in the DySS DNA polymerization system.
    • Fig. S8. Refueling experiments of the transient DySS DNA polymerization system.
    • Fig. S9. Average chain length in the transient DySS DNA polymerization system in dependence of the concentration of the DNA monomer M1.
    • Fig. S10. Characterization of the FRET duplex F and its cleaved and religated DNA fragments as used for in situ modulation of the DySS.
    • Fig. S11. ATP-dependent temporal control of the dynamic DNA bond with transient DySS FRET duplex formation.
    • References (40, 41)

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