Molecular robotic agents that survey molecular landscapes for information retrieval.

DNA-based artificial motors have allowed the recapitulation of biological functions and the creation of new features. Here, we present a molecular robotic system that surveys molecular environments and reports spatial information in an autonomous and repeated manner. A group of molecular agents, termed 'crawlers', roam around and copy information from DNA-labeled targets, generating records that reflect their trajectories. Based on a mechanism that allows random crawling, we show that our system is capable of counting the number of subunits in example molecular complexes. Our system can also detect multivalent proximities by generating concatenated records from multiple local interactions. We demonstrate this capability by distinguishing colocalization patterns of three proteins inside fixed cells under different conditions. These mechanisms for examining molecular landscapes may serve as a basis towards creating large-scale detailed molecular interaction maps inside the cell with nanoscale resolution.

In vitro selection of a trans aptamer complex for target-responsive fluorescence activation.

BACKGROUND: Most biological molecular complexes consist of multiple functional domains, yet rationally constructing such multifunctional complexes is challenging. Aptamers, the nucleic acid-based functional molecules, can perform multiple tasks including target recognition, conformational changes, and enzymatic activities, while being chemically synthesizable and tunable, and thus provide a basis for engineering enhanced functionalities through combination of multiple units. However, the conventional approach of simply combining aptamer units in a serial manner is susceptible to undesired crosstalk or interference between the aptamer units and to false interactions with non-target molecules; besides, the approach would require additional mechanisms to separate the units if they are desired to function independently. It is clearly a challenge to develop multi-aptamer complexes that preserve independent functions of each unit while avoiding undesired interference and non-specific interactions. RESULTS: By directly in vitro selecting a 'trans' aptamer complex, we demonstrate that one aptamer unit ('utility module') can remain hidden or 'inactive' until a target analyte triggers the other unit ('sensing module') and separates the two aptamers. Since the operation of the utility module occurs free from the sensing module, unnecessary crosstalk between the two units can be avoided. Because the utility module is kept inactive until separated from the complex, non-specific interactions of the hidden module with noncognate targets can be naturally prevented. In our demonstration, the sensing module was selected to detect serotonin, a clinically important neurotransmitter, and the target-binding-induced structure-switching of the sensing module reveals and activates the utility module that turns on a fluorescence signal. The aptamer complex exhibited a moderately high affinity and an excellent specificity for serotonin with ∼16-fold discrimination against common neurotransmitter molecules, and displayed strong robustness to perturbations in the design, disallowing nonspecific reactions against various challenges. SIGNIFICANCE: This work represents the first example of a trans aptamer complex that was in vitro selected de novo. The trans aptamer complex selected by our strategy does not require chemical modifications or immediate optimization processes to function, because the complex is directly selected to perform desired functions. This strategy should be applicable to a wide range of functional nucleic acid moieties, which will open up diverse applications in biosensing and molecular therapeutics.

Aptasensor-encapsulating semi-permeable proteinosomes for direct target detection in non-treated biofluids.

Detecting biomarkers in biofluids directly without sample treatments makes molecular diagnostics faster and more efficient. Aptasensors, the nucleic acid-based molecular biosensors, can detect a wide range of target molecules, but their susceptibility to degradation and aggregation by nucleases and charged proteins, respectively, limits their direct use in clinical samples. In this work, we demonstrate that when aptasensors are encapsulated in proteinosomes, the protein-based liposome mimics, clinically important small molecules can be sensitively and selectively detected in non-treated specimens, such as 100 % unpurified serum. As serum albumin is used to form the membrane, the nanomeshed proteinosomes become semi-permeable and antifouling, which enables exclusive admission of small molecules while blocking unwanted large proteins. Consequently, the enclosed aptasensors can maintain close-to-optimal performance for target binding, and nucleolytic degradation and electrostatic aggregation are effectively suppressed. Three different structure-switching aptamers specific for estradiol, dopamine, and cocaine, respectively, are demonstrated to fully conserve their high affinities and specificities inside the microcapsules. The shielding effect of proteinosomes is indeed exceptional; the enclosed DNA aptasensors remain completely intact over 18 h in serum and even in an extremely concentrated DNase solution (1 mg/ml, ∼300,000× the serum level). Moreover, the proteinosome-mediated compartmentalization enables independent operation of multiple aptasensors in the same mixture. Hence, simultaneous real-time sensing of two different targets is demonstrated with different operation modes, 'recording' target appearance and 'reporting' target concentration changes. This work is the first demonstration of small-molecule-specific aptasensors operating with optimal performance in serum environments and will find promising applications in molecular diagnostics.

Force Mapping Reveals the Spatial Distribution of Individual Proteins in a Neuron.

Conventional methods for studying the spatial distribution and expression level of proteins within neurons have primarily relied on immunolabeling and/or signal amplification. Here, we present an atomic force microscopy (AFM)-based nanoscale force mapping method, where Anti-LIMK1-tethered AFM probes were used to visualize individual LIMK1 proteins in cultured neurons directly through force measurements. We observed that the number density of LIMK1 decreased in neuronal somas after the cells were depolarized. We also elucidated the spatial distribution of LIMK1 in single spine areas and found that the protein predominantly locates at heads of spines rather than dendritic shafts. The study demonstrates that our method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons without signal amplification or labeling. We expected that this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.

Nanoscale Force-Mapping-Based Quantification of Low-Abundance Methylated DNA.

Methylation changes at cytosine-guanine dinucleotide (CpG) sites in genes are closely related to cancer development. Thus, detection and quantification of low-abundance methylated DNA is critical for early diagnosis. Here, we report an atomic force microscopy (AFM)-based quantification method for DNA that contains methyl-CpG at a specific site, without any treatment to the target DNA such as chemical labeling, fluorescence tagging, or amplification. We employed AFM-tip-tethered methyl-CpG-binding proteins to probe surface-captured methylated DNA. We observed a linear correlation (R2 = 0.982) between the input copy number and detected copy number, in the low copy number regime (10 or fewer; subattomolar concentrations). For a mixture of methylated and nonmethylated DNA that resembles clinical samples, we were still able to quantify the methylated DNA. These results highlight the potential of our force-mapping-based quantification method for wide applications in early detection of diseases associated with methylated DNA.

A DNA nanoscope via auto-cycling proximity recording.

Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to acquire a comprehensive set of pairwise proximities between components would satisfy an increasing interest in investigating individual macromolecules and their interactions, but current biochemical techniques detect only a single proximity partner per probe. Here, we present a biochemical DNA nanoscopy method that records nanostructure features in situ and in detail for later readout. Based on a conceptually novel auto-cycling proximity recording (APR) mechanism, it continuously and repeatedly produces proximity records of any nearby pairs of DNA-barcoded probes, at physiological temperature, without altering the probes themselves. We demonstrate the production of dozens of records per probe, decode the spatial arrangements of 7 unique probes in a homogeneous sample, and repeatedly sample the same probes in different states.The spatial organisation of nanostructures is fundamental to their function. Here, the authors develop a non-destructive, proximity-based method to record extensive spatial organization information in DNA molecules for later readout.

Self-assembly of two-dimensional DNA origami lattices using cation-controlled surface diffusion.

DNA origami has proven useful for organizing diverse nanoscale components into patterns with 6 nm resolution. However for many applications, such as nanoelectronics, large-scale organization of origami into periodic lattices is desired. Here, we report the self-assembly of DNA origami rectangles into two-dimensional lattices based on stepwise control of surface diffusion, implemented by changing the concentrations of cations on the surface. Previous studies of DNA­mica binding identified the fractional surface density of divalent cations (ñ(s2))as the parameter which best explains the behaviour of linear DNA on mica. We show that for ñ(s2) between 0.04 and 0.1, over 90% of DNA rectangles were incorporated into lattices and that, compared with other functions of cation concentration, ñ(s2) best captures the behaviour of DNA rectangles. This work shows how a physical understanding of DNA­mica binding can be used to guide studies of the higher-order assembly of DNA nanostructures, towards creating large-scale arrays of nanodevices for technology.

Effect of myopia and age on optic disc margin anatomy within the parapapillary atrophy area.

PURPOSE: To evaluate the effect of myopia and age on temporal optic disc margin anatomy within the parapapillary atrophy (PPA) area by use of spectral-domain optical coherence tomography (OCT). METHODS: Fifty young myopic eyes with PPA (myopic PPA group), 50 aged non-myopic eyes with PPA (aged PPA group), and 50 young non-myopic eyes without PPA (control group) were enrolled. High-definition OCT scanning was used to obtain horizontal cross-sectional optic nerve head (ONH) images. By use of these OCT scans we investigated three temporal optic disc margin structures: the configuration of the border tissue of Elschnig; the cross-sectional ONH structure coinciding with the clinically detected optic disc margin; and the integrity of the retinal layers within the PPA area. RESULTS: The distribution of the configuration of the border tissue of Elschnig and the cross-sectional ONH structure coinciding with the clinically detected optic disc margin of the myopic PPA group differed significantly from those of the control group (P < 0.01) whereas those of the aged PPA group did not (P > 0.05). Other than the photoreceptor layer, the retinal layers within the PPA area were more commonly impaired in the myopic PPA group than in the aged PPA group (P < 0.001). CONCLUSIONS: Myopia and aging led to different structural changes in temporal optic disc margin anatomy within the PPA area. This finding implies that different mechanisms may underlie myopic and age-related PPA development.

Programmable molecular recognition based on the geometry of DNA nanostructures.

From ligand-receptor binding to DNA hybridization, molecular recognition plays a central role in biology. Over the past several decades, chemists have successfully reproduced the exquisite specificity of biomolecular interactions. However, engineering multiple specific interactions in synthetic systems remains difficult. DNA retains its position as the best medium with which to create orthogonal, isoenergetic interactions, based on the complementarity of Watson-Crick binding. Here we show that DNA can be used to create diverse bonds using an entirely different principle: the geometric arrangement of blunt-end stacking interactions. We show that both binary codes and shape complementarity can serve as a basis for such stacking bonds, and explore their specificity, thermodynamics and binding rules. Orthogonal stacking bonds were used to connect five distinct DNA origami. This work, which demonstrates how a single attractive interaction can be developed to create diverse bonds, may guide strategies for molecular recognition in systems beyond DNA nanostructures.

"Fingertip"-guided noncovalent functionalization of carbon nanotubes by dendrons.

Noncovalent functionalization of carbon nanotubes (CNTs) by dendrons was demonstrated. Certain types of dendrons successfully functionalized CNT surfaces through the noncovalent interactions between the peripheries of the dendrons and the sidewalls of CNTs. Dendrons have a unique anisotropic shape and an orthogonal functional group at their apex, and thus can generate a certain spacing between the functional groups upon immobilization on surfaces. Atomic force microscope (AFM) imaging, dispersion experiments, and MicroRaman spectroscopy were employed for the characterization of the functionalization. The binding was found to be governed by the chemical nature of the terminal groups, namely, the "fingertips", through a comparison study on the adsorption efficiency of the dendron analogs. Functional groups such as the carboxylic acid group and the benzyl amide group were effective for the cooperative binding. AFM analysis showed that the average spacing generated by the dendrons was 14-15 nm at a particular adsorption condition. Assembling streptavidin on the tubes through the dendrons and biotin confirmed the realization of the regulated spacing as well as the elimination of unwanted aggregation. The noncovalent functionalization of CNTs by a dendron can be a new approach toward sensible nanobiodevices, not only by introducing biomolecular probes on CNTs without disruption of the electronic network of the tubes, but also by providing the immobilized probe molecules with a space ample enough to minimize steric hindrance for the unhindered interaction with their target species.