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.
Department of Chemistry, Metcalf Center for Science and Engineering, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Lauren K
LK
Wolf
Yang
Y
Gao
Rosina M
RM
Georgiadis
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