Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements.
We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.
Wolf
Lauren K
LK
Department of Chemistry, Metcalf Center for Science and Engineering, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Gao
Yang
Y
Georgiadis
Rosina M
RM
D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D017186:Titrimetry
D004364:Pharmaceutical Preparations; D012996:Solutions; D004247:DNA