Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems.

Interfacial Molecular Searching Using Forager Dynamics.

Many biological and technological systems employ efficient non-Brownian intermittent search strategies where localized searches alternate with long flights. Coincidentally, molecular species exhibit intermittent behavior at the solid-liquid interface, where periods of slow motion are punctuated by fast flights through the liquid phase. Single-molecule tracking was used here to observe the interfacial search process of DNA for complementary DNA. Measured search times were qualitatively consistent with an intermittent-flight model, and ∼10 times faster than equivalent Brownian searches, suggesting that molecular searches for reactive sites benefit from similar efficiencies as biological organisms.

Structure-Specific Liquid Crystal Anchoring Induced by the Molecular Combing of Short Oligonucleotides.

Surface-immobilized oligonucleotides were "combed" by meniscus motion and exposed to a nematic liquid crystal (LC). Although the oligonucleotides were as short as 16 bases, they were apparently oriented by this process and, in turn, successfully biased the orientation of the adjacent LC material. Single-stranded DNA (ssDNA) induced LC orientation in the combing direction, while hybridized double-stranded DNA (dsDNA) rotated the azimuthal LC orientation by ∼30° from the combing direction. The sensitivity of the chiral response to mixed ssDNA/dsDNA surfaces was characterized by employing complementary DNA that was longer than the immobilized DNA, resulting in single-stranded overhangs of various lengths. A rotated LC orientation was observed even when more than 70% of the DNA was single-stranded, and the transition from the rotated to nonrotated response was apparently discontinuous as a function of ssDNA surface coverage. These phenomena represent a sensitive DNA hybridization detection strategy that can potentially comprise a multiplexed assay.

Mechanisms of surface-mediated DNA hybridization.

Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100,000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state.

Effects of molecular size and surface hydrophobicity on oligonucleotide interfacial dynamics.

Single-molecule total internal reflection fluorescence microscopy was used to observe the dynamic behavior of polycytosine single-stranded DNA (ssDNA) (1-50 nucleotides long) at the interface between aqueous solution and hydrophilic (oligoethylene glycol-modified fused silica, OEG) and hydrophobic (octadecyltriethoxysilane-modified fused silica, OTES) solid surfaces. High throughput molecular tracking was used to determine >75,000 molecular trajectories for each molecular length, which were then used to calculate surface residence time and squared displacement (i.e., "step-size") distributions. On hydrophilic OEG surfaces, the surface residence time increased systematically with ssDNA chain length, as expected due to increasing molecule-surface interactions. Interestingly, the residence time decreased with increasing ssDNA length on the hydrophobic OTES surface, particularly for longer chains. Similarly, the interfacial mobility of polynucleotides slowed with increasing chain length on OEG, but became faster on OTES. On OTES surfaces, the rates associated with desorption and surface diffusion exhibited the distinctive anomalous temperature dependence that is characteristic of hydrophobic interactions for short-chain species but not for longer chains. These combined observations suggest that long oligonucleotides adopt conformations minimizing hydrophobic interactions, e.g., by internal sequestration of hydrophobic nucleobases.

Detecting nanoparticulate silver using single-particle inductively coupled plasma-mass spectrometry.

The environmental prevalence of engineered nanomaterials, particularly nanoparticulate silver (AgNP), is expected to increase substantially. The ubiquitous use of commercial products containing AgNP may result in their release to the environment, and the potential for ecological effects is unknown. Detecting engineered nanomaterials is one of the greatest challenges in quantifying their risks. Thus, it is imperative to develop techniques capable of measuring and characterizing exposures, while dealing with the innate difficulties of nanomaterial detection in environmental samples, such as low-engineered nanomaterial concentrations, aggregation, and complex matrices. Here the authors demonstrate the use of inductively coupled plasma-mass spectrometry, operated in a single-particle counting mode (SP-ICP-MS), to detect and quantify AgNP. In the present study, two AgNP products were measured by SP-ICP-MS, including one of precisely manufactured size and shape, as well as a commercial AgNP-containing health food product. Serial dilutions, filtration, and acidification were applied to confirm that the method detected particles. Differentiation of dissolved and particulate silver (Ag) is a feature of the technique. Analysis of two wastewater samples demonstrated the applicability of SP-ICP-MS at nanograms per liter Ag concentrations. In this pilot study, AgNP was found at 100 to 200 ng/L in the presence of 50 to 500 ng/L dissolved Ag. The method provides the analytical capability to monitor Ag and other metal and metal oxide nanoparticles in fate, transport, stability, and toxicity studies using a commonly available laboratory instrument. Rapid throughput and element specificity are additional benefits of SP-ICP-MS as a measurement tool for metal and metal oxide engineered nanoparticles.