Synthesis and Direct Observation of Thermoresponsive DNA Copolymers

Single-molecule techniques allow for the direct observation of long-chain macromolecules, and these methods can provide a molecular understanding of chemically heterogeneous and stimuli-response polymers. In this work, we report the synthesis and direct observation of thermoresponsive DNA copolymers using single-molecule techniques. DNA-PNIPAM copolymers are synthesized using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing non-natural nucleotides (dibenzocyclooctyne-dUTP), followed by grafting thermoresponsive side branches (poly(N-isopropylacrylamide), PNIPAM) onto DNA backbones using copper-free click chemistry. Single-molecule fluorescence microscopy is used to directly observe the stretching and relaxation dynamics of DNA-PNIPAM copolymers both below and above the lower critical solution temperature (LCST) of PNIPAM. Our results show that the intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by temperature, branch density, and branch molecular weight. Single-molecule experiments reveal an underlying molecular heterogeneity associated with polymer stretching and relaxation behavior, which arises in part due to heterogeneous chemical identity on DNA copolymer dynamics. T chemical composition of long-chain macromolecules plays a key role in determining the emergent physical and structural properties of polymeric systems. Copolymers and block polymers with simple linear chain architectures can give rise to intricate structural ordering and assembly across multiple length scales. In addition to chemical identity, polymer chain architecture also plays a key role in supramolecular assembly, with chain topologies and shapes including linear, branched, ring, and cross-linked polymers. Synthetic strategies for controlling polymer composition and architecture have been used to develop materials for applications including encapsulation for drug delivery, gene delivery, catalysis, and surfactants for emulsion stabilization. In recent years, biohybrid materials containing both synthetic and natural components have been developed, which has provided intriguing new materials with increased biocompatibility, controlled biodegradation, broad chemical functionality, and unprecedented levels of sequence control. In particular, DNA copolymers have been shown to self-assemble into various supramolecular architectures such as micelles, vesicles, and tubular structures. Despite recent work, however, we still lack a full understanding of the effects of chemistry, architecture, and hydrophilicity/hydrophobicity on the conformational dynamics of DNA-based copolymers. Single-molecule techniques offer an ideal strategy to directly study the conformational dynamics of macromolecules. In recent years, DNA has served as a model polymer for fundamental studies of nonequilibrium polymer dynamics ranging from dilute to entangled polymer solutions. However, the vast majority of prior single polymer studies has largely focused on dynamic studies of chemically homogeneous, linear DNA. Recent work has extended the field of single polymer dynamics to chains with nonlinear architectures including ring polymers and comb polymers, though these macromolecules generally consist of natural DNA. On the other hand, molecular assembly of DNA-based materials has been studied using bulk spectroscopic methods. However, the conformational dynamics of chemically heterogeneous copolymers has not yet been fully explored at the single-molecule level. From this view, development of new methods to directly image the triggered response or assembly of functional copolymers at the single-molecule level would provide new insights into the molecular-scale dynamics of these materials. For example, direct imaging of the thermally Received: January 5, 2018 Accepted: February 11, 2018 Letter pubs.acs.org/macroletters Cite This: ACS Macro Lett. 2018, 7, 281−286 © XXXX American Chemical Society 281 DOI: 10.1021/acsmacrolett.8b00016 ACS Macro Lett. 2018, 7, 281−286 activated response of DNA-PNIPAM copolymers at the singlechain level could reveal molecular distributions and unexpected heterogeneous behavior, thereby providing new information beyond traditional bulk cloud point measurements. In this work, we report the synthesis and direct observation of thermoresponsive DNA copolymers consisting of a main chain DNA backbone with grafted poly(N-isopropylacrylamide) (PNIPAM) side branches. PNIPAM is a thermoresponsive polymer exhibiting a reversible hydrophilic to hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST) (32 °C). From this perspective, PNIPAM is a suitable material to generate DNA copolymers with temperature-sensitive properties near or slightly above room temperature. Following synthesis and characterization, single-molecule fluorescence microscopy is used to observe the conformational stretching and relaxation dynamics of single DNA-PNIPAM copolymers both below and above the LCST of PNIPAM. Our results show that the hydrophilic−hydrophobic transition for PNIPAM side branches results in enhanced intramolecular interactions, leading to dynamic heterogeneity in single polymer behavior. In particular, experiments reveal broadly distributed probabilities of polymer chain extension above the LCST and altered conformational relaxation dynamics due to PNIPAM side branches. Taken together, these results demonstrate the utility of studying the dynamics of biohybrid copolymers using single-molecule