We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.

We present results of the first systematic study on in situ sequence-dependent kinetics for short singlestrand oligonucleotide surface immobilization. By measuring film coverage for both thiolated and nonthiolated homo-oligomers as a function of adsorption time, we determine the relative contribution of specific thiol-surface and nonspecific DNA-surface interactions to the overall mechanism of DNA-thiol attachment to gold. We find that sequence-dependent nonspecific surface interactions play a significant role in DNA-thiol immobilization, influencing not only the kinetics but also the extent of oligomer adsorption. For example, sequences that initially form strong, rapid nonspecific contacts with the surface hinder long-time thiol adsorption (i.e., poly(dA)-thiol). In contrast, sequences with nucleotides that initially bind slowly and weakly to the surface (i.e., poly(dT)-thiol) do not obstruct further thiol adsorption, resulting in higher film coverage and Langmuir immobilization kinetics. This view of the DNA-thiol immobilization mechanism is further supported by sequence-dependent rinsing losses observed for thiolated DNA strands but not for analogous nonthiolated strands. Nonthiolated strands contact the surface strongly in a more horizontal orientation, whereas thiolated strands attain a more upright orientation that allows vertical DNA-DNA base-stacking. The results clearly illustrate the importance and interplay of competitive specific and nonspecific forces in forming DNA-thiol films. The specific coverage attained and the time dependence of the adsorption process depend on the prevailing sequence composition.

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.

We demonstrate the quantitative characterization of DNA-DNA and DNA-drug interactions by angle-resolved surface plasmon resonance (SPR) imaging. Combining the angle-scanning capabilities of traditional SPR with the spatial definition capabilities of imaging, we directly measure DNA and drug surface coverages and kinetics simultaneously for multiple patterned spots. We find excellent agreement of DNA-DNA hybridization kinetics and thermodynamics measured by both the imaging system and traditional SPR. Instrument response and sensitivity is further demonstrated by successful measurement of association and dissociation kinetics of actinomycin-D binding to a low-density doubled-stranded DNA binding sequence. Without independent calibration, analysis of angle-resolved SPR imaging data yields 2.9 +/- 0.1 drugs per duplex at saturation coverage, consistent with all available duplex binding sites being occupied.

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.