Single Molecule Characterization of Dual-Colored DNA Comb Polymers

Single Molecule Characterization of Dual-Colored DNA Comb Polymers

Danielle J. Mai, Amanda B. Marciel, Charles M. Schroeder

We report the synthesis and single molecule characterization of two-color, comb-shaped branched biopolymers. In this work, we utilize a hybrid enzymatic-synthetic approach to graft “short” DNA branches to “long” DNA backbones, thereby producing macromolecular DNA comb polymers (Figure 1). The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs) and primers: “short” branches consist of internal Cy5 labels and a terminal azide group, and “long” backbones contain internal dibenzylcyclooctyne (DBCO) groups and a terminal biotin tag. In this way, we utilize strain-promoted, Cu(I)-free [2+3] cycloaddition “click” chemistry for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, resulting comb polymers can be labeled with a DNA stain (e.g., SYTOX Green®) for dual-color imaging. To this end, we use single molecule fluorescence microscopy to directly observe comb polymers by tethering probe molecules to a surface with biotin-NeutrAvidin linkages, extending them with pressure-driven flow, and imaging them with fluorescence microscopy (Figure 2). Our imaging approach allows for characterization of these materials at the single molecule level (e.g., quantification of polymer contour length and branch distributions for varying synthetic conditions). We are also extending this approach to single polymer rheology experiments, with specific aims to understand the effects of branching on relaxation timescales (surface tethered or free solution), steady-state extension in various flow fields, and dynamics in semi-dilute or concentrated solutions. In this way, our work will contribute to the overall understanding of topologically complex polymer melts and solutions.

 

Figure 1. Synthesis of biotin-tagged, dual-color DNA comb polymer molecules.

Figure 2. Single molecule images of tethered dual-color DNA comb extension under shear flow at a surface. (a) Co-localized composite image; (b) SYTOX Green-stained backbones and branches; (c) Cy5-labeled branches.