Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

Abstract

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA.ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA.ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA.ligand complexes.

Melting Thermodynamics of Reversible DNA/Ligand Complexes at Interfaces

“…Other approaches have relied on tagging the small molecule with a fluorophore, 22–23 or on monitoring binding through the force required to separate the two strands of a duplex in the presence of the small molecule. 24 Many of these advances have exploited the excellent high-throughput capacity, and frugal reagent consumption, of microarrayed supports.…”

Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands

“…In contrast, providing the ligand-binding sequence with a spacer and locating away from the surface by implementing it in the upper RNA duplex yielded reliable values for the dissociation constant in agreement with literature values. Although the dissociation constants measured by other more laborious and time-consuming techniques might be more accurate, our assay provides sufficiant accuracy in a parallel screening format for dissociation constants, ranging from the picomolar (26) (chiral polyamides binding to DNA) to the high micromolar scale (23) (DNA-aptamer specific for ATP). Moreover, the current format with 16 spots can be varied to titrate two ligands in parallel (eight spots per ligand) or change the binding sequence in half the spots in order to gain a deeper insight into the ligand-binding sequence interaction in a one-shot experiment.…”

“…The normalized fluorescence is given by . A detailed description can be found in (26). The normalized fluorescence is thus the fraction of intact lower bonds of the total number of molecules under load.…”

A high throughput molecular force assay for protein–DNA interactions

“…The detailed derivation for the NF has been given previously. 24 In short: initially, all molecular setups are present in the state S0 and were detected via the Cy5 labeled oligomer 2 ( Fig. 1(a1)).…”