Characterizing functional
integrity of the APH construct. (A) Dynamic
light scattering measurements revealed a narrow radius distribution
of ∼10 nm with negligible aggregation in phosphate-buffered
saline buffer. (B) Gel electrophoresis shows that APH conjugation
via (BimC4A)3 ligand-accelerated CuAAC greatly
minimizes DNA damage. Lanes: 1, unconjugated polymer; 2, unconjugated
aptamer (45 nt); 3, APH synthesized with (Bim)3; 4, APH
synthesized with (BimC4A)3; 5, 20-bp ladder.
(C) Affinity measurements from a bead-based nucleolin-binding assay
show that aptamer target affinity is preserved within the APH construct.
The unconjugated aptamer exhibits a Kd of 8.37 ± 0.75 nM (black), while APH molecules consisting of
Cy5-labeled aptamer and coumarin-labeled polymer scaffold display
similar Kds of 5.18 ± 0.72 and 4.01
± 0.72 nM based on Cy5 (red) and coumarin (blue) intensities,
respectively. In contrast, unconjugated polymer (gray) exhibits negligible
nucleolin affinity. (D) Time-dependent fluorescence measurements confirm
selective payload release. Untreated APHs emit a red-shifted, self-quenched
signal (red), but esterase treatment shifts the peak fluorescence
wavelength to that of free coumarin (blue), with a signal that increases
over time as more coumarin is released (dashed lines).