Reversible strain-promoted DNA polymerization

Abstract

Molecular strain can be introduced to influence the outcome of chemical reactions. Once a thermodynamic product is formed, however, reversing the course of a strain-promoted reaction is challenging. Here, a reversible, strain-promoted polymerization in cyclic DNA is reported. The use of nonhybridizing, single-stranded spacers as short as a single nucleotide in length can promote DNA cyclization. Molecular strain is generated by duplexing the spacers, leading to ring opening and subsequent polymerization. Then, removal of the strain-generating duplexers triggers depolymerization and cyclic dimer recovery via enthalpy-driven cyclization and entropy-mediated ring contraction. This reversibility is retained even when a protein is conjugated to the DNA strands, and the architecture of the protein assemblies can be modulated between bivalent and polyvalent states. This work underscores the utility of using DNA not only as a programmable ligand for assembly but also as a route to access restorable bonds, thus providing a molecular basis for DNA-based materials with shape-memory, self-healing, and stimuli-responsive properties.

Fig. 1.. Schematic showing the strain-promoted DNA polymerization.

(A) Polymerization reaction after duplexing the nonhybridizing, single-stranded regions of cyclic dimers. (B) Conceptual overall free energy diagram. (C) Pathways of shape deformation and recovery after the removal of the strain-generating duplexers.

Fig. 2.. Synthesis and characterization of cyclic dimers.

(A) Reaction scheme showing monomers (top) with single-stranded DNA spacers (gray) forming linear dimers via the initial association between one pair of sticky ends (middle). Subsequent cyclization (bottom) is expected to be favored over polymerization due to the flexible spacer. (B) Polyacrylamide (12%) gel electrophoresis of unpurified reaction mixtures and linear dimers with 0-, 1-, and 14-nt spacers. Triangular arrows indicate the major product. (C) Analytical size exclusion chromatogram of reaction mixtures, linear dimers, and monomers with 1- and 14-nt spacers. (D) Quantification of cyclic dimers across different spacer lengths and sequences. (E) Calculated elastic energies of cyclic dimers as a function of spacer length. (F) AFM image of the reaction with 0-nt spacers. (G) AFM image of the reaction with 14-nt spacers along with a detailed view of a single cyclic dimer (inset). Scale bar, 10 nm (inset). (H) Measured bending angles and the percentage of DNA bubble formation observed using AFM.

Fig. 3.. Reversible strain-promoted polymerization.

(A) Reaction scheme showing the addition of complementary duplexers to cyclic dimers containing spacers, leading to strain-promoted ring opening and polymerization. Subsequent removal of the strain-generating duplexers using toehold-mediated strand displacement drives the reaction back to cyclic dimers. (B) Agarose (3%) gel electrophoresis of the polymerization and recovery process. Lane 1, starting cyclic dimers with 24-nt spacers; lane 2, polymerization products after the addition of duplexers to the reaction mixture; lane 3, regenerated cyclic dimers after duplexer strand displacement; lanes 4 and 5, mixtures of duplexers with their respective complementary displacement strands, equivalent to displaced duplexers. (C) AFM image of polymers after ring opening. (D) Quantification of polymer length and degree of polymerization. (E) Degree of polymerization with varying ratios of duplexers added. (F) Yields of cyclic dimers over multiple duplexer addition and displacement cycles.

Fig. 4.. Mechanistic studies of spontaneous depolymerization.

(A) Percent yield of cyclic dimers after incubating polymers without duplexers as a function of time at various temperatures. (B) Scheme of chain extension reaction using linear oligomers via the addition of single monomers. Linear oligomers with either single-stranded or duplexed spacers can be extended after adding a single monomer with single-stranded or duplexed spacers, respectively. For clarity, only single-stranded spacers are shown. (C) Agarose (2%) gel electrophoresis of the polymerization reaction (middle) and after the addition of capping monomers with duplexed spacers (right). (D) Agarose (2%) gel electrophoresis of the reaction with displaced duplexers after 5 min of incubation at 25°C (middle) and the same reaction after the addition of capping monomers with single-stranded spacers (right). (C and D) Cyclic structures are identified in positions where the band intensity is relatively unchanged before and after capping. Arrows indicate the linear chains with enriched intensities on gel. (E) Percentage of cyclic products incubated at 25°C, over the course of 27 hours. (F) Relative distribution among cyclic oligomers after 1 hour at various temperatures. (G) Relative distribution among cyclic oligomers at 45°C, over the course of 27 hours. (H) Proposed two-step depolymerization route involving cyclization and ring contraction.

Fig. 5.. Dynamic control of the oligomerization state and architectures of protein assemblies using DNA.

(A) Synthesis of protein-DNA conjugates via an amine- and thiol-reactive bifunctional crosslinker. (B) Scheme for the modulation of protein assembly via duplexer addition and strand displacement. (C) Analytical size exclusion chromatogram for different assembly states in (B). Dashed lines are guides for the eye. (D to F) AFM images of protein-DNA conjugates in (D) monomeric, (E) dimeric, and (F) polymeric states. Scale bars, 140 nm and 10 nm (insets). (G) Degree of polymerization of protein-DNA conjugates.