(98k) Flexible Branched Polymers for Single Molecule Rheology

In this work, we report the synthesis of well-characterized branched biopolymers for single molecule rheology. We extended the method of enzymatic polymerization of long linear polymers consisting of single-stranded DNA (ssDNA) to include bioorthogonal attachment chemistries, thereby enabling the synthesis of well-defined, branched ssDNA polymer molecules. The resulting polymers are suitable for single-molecule flow studies, which have potential to improve the development of molecular-based theories for topologically-complex polymer melts and solutions. Recently, our lab has pioneered the use of ssDNA as a new model system to study the dynamics of flexible polymer chains. ssDNA provides a superior alternative to double-stranded DNA (dsDNA), which has served as a model for single molecule polymer dynamics in flow for the past two decades. dsDNA is well known as a semi-flexible polymer with markedly different local molecular properties and a large persistence length (l ≈ 53 nm) compared to flexible polymer chains, such as polystyrene (l ≈ 0.7 nm) and ssDNA (l ≈ 0.6 nm). In prior work, we have successfully synthesized long, fluorescently labeled ssDNA chains, which we have studied in extensional flow with epifluorescence microscopy.

In the current study, we synthesize and characterize branched polymers based on ssDNA for single molecule visualization. We rely on enzymatic template-directed polymerization to incorporate a chemically modified deoxyribonucleotide at pre-determined locations along the backbone. We utilize the modified nucleotides as “grafting sites” for facile integration of azide-terminated single stranded DNA branches via copper-mediated alkyne-azide cycloaddition or strain-promoted Cu(I)-free [2+3] cycloaddition “click” reactions. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Using this approach, we have synthesized branched polymers with three arm star, H-shaped, and comb architectures. In future work, we will utilize a microfluidic-based, automated, hydrodynamic trap to observe various timescales of branched polymer behavior by varying and oscillating strain rates. Overall, we seek a molecular-based understanding of the non-equilibrium dynamics of flexible polymer chains, which is crucial for control in processing and molecular self-assembly.