Surface-Mediated DNA Hybridization: Effects of DNA Conformation, Surface Chemistry, and Electrostatics.

Single-molecule Förster Resonance Energy Transfer (FRET) was used to study the dynamic association of mobile donor-labeled ssDNA oligonucleotides ("target") with covalently immobilized complementary acceptor-labeled ssDNA oligonucleotides ("probe"). While probe-target association events were resolved for all experiments, such FRET events were far more likely to occur in systems with complementarity and on hydrophobic, as compared to hydrophilic, surfaces. The distribution of donor-acceptor association-time intervals did not exhibit simple first-order kinetics, and when decomposed into a superposition of first-order processes, only a small fraction of events corresponded to a long-lived state that was presumed to represent true DNA hybridization, while the majority of association events were transient, representing nonspecific associations or partial hybridization. The structure of the DNA target and probe affected both the stability of the hybridized state, as well as the likelihood that an association between the two led to hybridization. In particular, the likelihood of hybridization decreased for longer target strands and for targets with stem-loop secondary structure. The presence of oligonucleotide secondary structure reduced the stability of hybridization, while greater complementarity increased stability of the hybridized state. Interestingly, increased ionic strength (i.e., greater electrostatic screening) increased the probability of hybridization but did not influence the lifetime of the hybridized state. Combined, these observations provide a nuanced view of surface-mediated DNA hybridization, where various factors independently influence the probability and stability of hybridization.

Hydroporphyrin-Doped Near-Infrared-Emitting Polymer Dots for Cellular Fluorescence Imaging.

Near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) have shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. This paper describes the synthesis of NIR-emitting Pdots with great control and tunability of emission peak wavelength. The Pdots were prepared by doping poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)] (PFBT), a semiconducting polymer commonly used as a host polymer in luminescent Pdots, with a series of chlorins and bacteriochlorins with varying functional groups. Chlorins and bacteriochlorins are ideal dopants due to their high hydrophobicity, which precludes their use as molecular probes in aqueous biological media but on the other hand prevents their leakage when doped into Pdots. Additionally, chlorins and bacteriochlorins have narrow deep red to NIR-emission bands and the wide array of synthetic modifications available for modifying their molecular structure enables tuning their emission predictably and systematically. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements show the chlorin- and bacteriochlorin-doped Pdots to be nearly spherical with an average diameter of 46 ± 12 nm. Efficient energy transfer between PFBT and the doped chlorins or bacteriochlorins decreases the PFBT donor emission to near baseline level and increases the emission of the doped dyes that serve as acceptors. The chlorin- and bacteriochlorin-doped Pdots show narrow emission bands ranging from 640 to 820 nm depending on the doped dye. The paper demonstrates the utility of the systematic chlorin and bacteriochlorin synthesis approach by preparing Pdots of varying emission peak wavelength, utilizing them to visualize multiple targets using wide-field fluorescence microscopy, binding them to secondary antibodies, and determining the binding of secondary antibody-conjugated Pdots to primary antibody-labeled receptors in plant cells. Additionally, the chlorin- and bacteriochlorin-doped Pdots show a blinking behavior that could enable their use in super-resolution imaging methods like STORM.

Interplay of electrostatic repulsion and surface grafting density on surface-mediated DNA hybridization.

Single-molecule Förster Resonance Energy Transfer was used to observe the adsorption of fluorescently-labeled "target" DNA oligonucleotides and their association and hybridization with complementary DNA "probes" tethered to the surface as a function of surface grafting density. Ionic strength was varied systematically to disentangle the potentially competing effects of probe accessibility and electrostatic repulsion. At high ionic strength, when the Debye length was ~1 nm, the adsorption of target DNA was not significantly inhibited by the presence of tethered probe DNA, even at high grafting density, and the fraction of adsorbed target strands undergoing hybridization increased systematically with grafting density, leading to a dramatic increase in the net hybridization rate at high grafting density. However, at lower ionic strength, when the Debye length was ≥3 nm, the adsorption rate of target DNA decreased and the fraction of adsorbed target strands undergoing hybridization saturated at high probe grafting density (≥7,000 strands/µm2), presumably due to electrostatic repulsion. As a result, the net rate of hybridization exhibited a maximum as a function of grafting density. This has important consequences for the design of systems that optimize surface-mediated DNA hybridization under low-salt high-stringency conditions.

Influence of Oligonucleotide Grafting Density on Surface-Mediated DNA Transport and Hybridization.

Adsorption of soluble DNA to surfaces decorated with complementary DNA plays an important role in many bionanotechnology applications, and previous studies have reported complex dependencies of the surface density of immobilized DNA on hybridization. While these effects have been speculatively ascribed to steric or electrostatic effects, the influence of surface-mediated molecular transport (i.e., intermittent "hopping diffusion") has not been fully appreciated. Here, single-molecule tracking and Förster resonance energy transfer (FRET) were employed to characterize the mobility and the hybridization efficiency of adsorbed ssDNA oligonucleotides ("target") at solid-liquid interfaces exhibiting surface-immobilized ssDNA ("probe") over a wide range of surface grafting densities. Two distinct regimes were observed, with qualitatively different transport and hybridization behaviors. At dilute grafting density, only 1-3% of target molecules were observed to associate with probes (i.e., to hybridize). Adsorbing target molecules often searched unsuccessfully and "flew", via desorption-mediated diffusion, to secondary locations before hybridizing. In contrast, at high probe grafting density, approximately 20% of target DNA hybridized to immobilized probes, and almost always in the vicinity of initial adsorption. Moreover, following a dehybridization event, target molecules rehybridized at high probe density, but rehybridization was infrequent in the dilute density regime. Interestingly, the intermittent interfacial transport of mobile target molecules was suppressed by the presence of immobilized probe DNA, presumably due to an increased probability of readsorption following each "hop". Together, these findings suggested that many salient effects of grafting density on surface-mediated DNA hybridization can be directly related to the mechanisms of surface-mediated intermittent diffusion.