(226w) Single Molecule Characterization of Dual-Colored DNA Comb Polymers

Mai, D. J., University of Illinois at Urbana-Champaign
Marciel, A. B., University of Chicago
Schroeder, C. M., University of Illinois at Urbana-Champaign

text-align:center;line-height:normal"> font-family:"Times New Roman","serif"'>Single Molecule Characterization of Dual-Colored
DNA Comb Polymers

text-align:center;line-height:normal"> "Times New Roman","serif"'>Danielle J. Mai, Amanda B. Marciel, Charles M.


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.




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




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.