A split aptamer for adenosine triphosphate (ATP) was embedded as a recognition unit into two levers of a nanomechanical DNA origami construct by extension and modification of selected staple strands. An additional optical module in the stem of the split aptamer comprised two different cyanine-styryl dyes that underwent an energy transfer from green (donor) to red (acceptor) emission if two ATP molecules were bound as target molecule to the recognition module and thereby brought the dyes in close proximity. As a result, the ATP as a target triggered the DNA origami shape transition and yielded a fluorescence color change from green to red as readout. Conventional atomic force microscopy (AFM) images confirmed the topology change from the open form of the DNA origami in the absence of ATP into the closed form in the presence of the target molecule. The obtained closed/open ratios in the absence and presence of target molecules tracked well with the fluorescence color ratios and thereby validated the bicolor fluorescence readout. The correct positioning of the split aptamer as the functional unit farthest away from the fulcrum of the DNA origami was crucial for the aptasensing by fluorescence readout. The fluorescence color change allowed additionally to follow the topology change of the DNA origami aptasensor in real time in solution. The concepts of fluorescence energy transfer for bicolor readout in a split aptamer in solution, and AFM on surfaces, were successfully combined in a single DNA origami construct to obtain a bimodal readout. These results are important for future custom DNA devices for chemical-biological and bioanalytical purposes because they are not only working as simple aptamers but are also visible by AFM on the single-molecule level.

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DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications.

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Key message Size limits on molecular movement among female gametes. AbstractCellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

The arabino-configured analog of uridine with a propargyl group at the 2’-position was synthesized and incorporated into DNA by solid-phase chemistry. The fluorescence quantum yields of DNA strands that were postsynthetically modified by blue and green emitting cyanine-styryl dyes were improved due to the arabino-configured anchor. These oligonucleotides were used as energy transfer donors in hybrids with oligonucleotides modified with acceptor dyes that emit in the yellow-red range. These combinations give energy transfer pairs with blue–yellow, blue–red and green–red emission color changes. All combinations of arabino- and ribo-configured donor strands with arabino- and ribo-configured acceptor strands were evaluated. This array of doubly modified hybrids was screened by their emission color contrast and fluorescence quantum yield. Especially mixed combinations, that means donor dyes with arabino-configured anchor with acceptor dyes with ribo-configured anchor, and vice versa, showed significantly improved fluorescence properties. Those were successfully applied for fluorescent imaging of DNA after transport into living cells.

In this protocol, we demonstrate a method for the synthesis of 2'-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period. Video LinkThe video component of this article can be found at http://www.jove.com/video/54121/ ProtocolCaution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are toxic and carcinogenic. Please use all appropriate safety practices that are typically required in organic chemistry laboratories, such as wearing a laboratory coat, safety glasses and gloves. Synthesis of the DyesNote: Both dyes can be synthesized by the same types of reaction. Figure 2 shows an overview of these reactions. 1. Synthesis of 1-(3-azidopropyl)-4-(2-(1-methyl-1H-indol-3-yl)vinyl)pyridin-1-ium iodide (dye 1)