Nanoscale Spatial Distribution of Thiolated DNA on Model Nucleic Acid Sensor Surfaces

The nanoscale arrangement of the DNA probe molecules on sensor surfaces has a profound impact on molecular recognition and signaling reactions on DNA biosensors and microarrays. Using electrochemical atomic force microscopy, we have directly determined the nanoscale spatial distribution of thiolated DNA that are attached to gold <i>via</i> different methods. We discovered significant heterogeneity in the probe density and limited stability for DNA monolayers prepared by the backfilling method, that is, first exposing the surface to thiolated DNA then “backfilling” with a passivating alkanethiol. On the other hand, the monolayers prepared by “inserting” thiolated DNA into a preformed alkanethiol monolayer lead to a more uniformly distributed layer of DNA. With high-resolution images of single DNA molecules on the surface, we have introduced spatial statistics to characterize the nanoscale arrangement of DNA probes. The randomness of the spatial distribution has been characterized. By determining the local densities surrounding individual molecules, we observed subpopulations of probes with dramatically different levels of “probe crowding”. We anticipate that the novel application of spatial statistics to DNA monolayers can enable a framework to understand heterogeneity in probe spatial distributions, interprobe interactions, and ultimately probe activity on sensor surfaces

A Single-Molecule View of Conformational Switching of DNA Tethered to a Gold Electrode

Surfaces that can actively regulate binding affinities or catalytic properties in response to external stimuli are a powerful means to probe and control the dynamic interactions between the cell and its microenvironment. Active surfaces also enable novel functionalities in biosensors and biomolecular separation technologies. Although electrical stimuli are often appealing due to their speed and localization, the operation of these electrically activated surfaces has mostly been characterized with techniques averaging over many molecules. Without a molecular-scale understanding of how biomolecules respond to electric fields, achieving the ultimate detection sensitivity or localized biological perturbation with the ultimate resolution would be difficult. Using electrochemical atomic force microscopy, we are able to follow the conformational changes of individual, short DNA molecules tethered to a gold electrode in response to an applied potential. Our study reveals conformations and dynamics that are difficult to infer from ensemble measurements: defects in the self-assembled monolayer (SAM) significantly perturb conformations and adsorption/desorption kinetics of surface-tethered DNA; on the other hand, the SAM may be actively molded by the DNA at different potentials. These results underscore the importance of characterizing the systems at the relevant length scale in the development of electrically switchable biofunctional surfaces

Electric-Field Dependent Conformations of Single DNA Molecules on a Model Biosensor Surface

Despite the variety of nucleic acid sensors developed, we still do not have definite answers to some questions that are important to the molecular binding and, ultimately, the sensitivity and reliability of the sensors. How do the DNA probes distribute on the surface at the nanoscale? As the functionalized surfaces are highly heterogeneous, how are the conformations affected when the probe molecules interact with defects? How do DNA molecules respond to electric fields on the surface, which are applied in a variety of detection methods? With in situ electrochemical atomic force microscopy and careful tailoring of nanoscale surface interactions, we are able to observe the nanoscale conformations of individual DNA molecules on a model biosensor surface: thiolated DNA on a gold surface passivated with a hydroxyl-terminated alkanethiol self-assembled monolayer. We find that under applied electric fields, the conformations are highly sensitive to the choice of the alkanethiol molecule. Depending on the monolayer and the nature of the defects, the DNA molecules may either adopt a highly linear or a highly curved conformation. These unusual structures are difficult to observe through existing “ensemble” characterizations of nucleic acid sensors. These findings provide a step toward correlating target-binding affinity, selectivity, and kinetics to the nanoscale chemical structure of and around the probe molecules in practical nucleic acid devices

Electrochemical Nanoscale Templating: Laterally Self-Aligned Growth of Organic–Metal Nanostructures

The electrodeposition of Ag into organized surfactant templates adsorbed onto (22 × √3) reconstructed Au(111) is investigated by in situ electrochemical scanning tunneling microscopy. Ag⁺ concentrations of as low as 2.5 × 10^–6 M allow the visualization of the electrochemical molecular templating effect of a sodium dodecyl sulfate (SDS) adlayer. The SDS hemicylindrical stripes determine the adsorption sites of the Ag⁺ ions and the directionality of Ag nanodeposition. The SDS-Ag nanostructures grow along the long axis of SDS hemicylindrical stripes, and an interaction of Ag with the Au(111) substrate leads to a structural change in the SDS stripe pattern. The SDS-Ag nanostructures undergo dynamic rearrangement in response to changes in the applied electrode potential. At negative potentials, the orientations of SDS-Ag nanostructures are pinned by the (22 × √3) reconstructed pattern. Furthermore, observed differences in Ag nanostructuring on Au(111) without molecular templates (i.e., on a bare Au(111) surface) confirm the role of self-assembled organic templates in producing metal–organic nanostructures under control of the surface potential, which can determine the feature size, shape, and period of the metal nanostructure arrays

(Related to S6 Fig)- <i>prdx-2</i> mutant PHA response to 10μM H₂O₂.

On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig. (MP4)</p

(Related to Fig 3)- Wild-type I2 response to 10μM H₂O₂.

On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a control animal. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig. (MP4)</p

(Related to Fig 4)- <i>sek-1</i> mutant PHA response to 10μM H₂O₂.

On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig. (MP4)</p

Related to Fig 4- <i>gur-3</i> and <i>lite-1</i> mutants show reciprocal phenotypes in H₂O₂ sensing in I2 and in PHA neurons.

(A-D) Average curves showing the normalized calcium response to 1mM H2O2 measured over time (indicated in seconds) using the GCaMP3 sensor in I2 and PHA left and right neurons (top and bottom curves) in gur-3(ok2245) (A,B) and lite-1(ce314) mutants (C,D). N, number of movies analyzed for each genotype. See S8–S11 Movies and related S8 Fig. (TIF)</p

Total Synthesis and Stereochemical Assignment of Callyspongiolide

Total synthesis of four callyspongiolide stereoisomers led to unambiguous assignment of relative and absolute stereochemistry of the natural product. Key features of the convergent, fully stereocontrolled route include the use of Krische allylation, Kiyooka Aldol reaction, Kociénski–Julia olefination, Still–Gennari olefination, Yamaguchi macrocyclization, and Sonogashira coupling reaction. Biological evaluation of the synthesized compounds against an array of cancer cells revealed that the stereochemistry of the macrolactone core played an important role

S14 Fig -

(TIF)</p