Synthesis of DNA Modified with Boronic Acid: Compatibility to Copper(I)-Catalyzed Azide-Alkyne Cycloaddition.

The postsynthetic and sequence-specific ligation chemistry of a phenylboronic acid to oligonucleotides using the amide bond formation was worked out. In the first coupling experiments with 4-carboxyphenylboronic acid, a 5'-hexylamino-modified oligonucleotide was used to evaluate and optimize the reaction conditions. This postsynthetic modification works best in the presence of TBTU and triethanolamine and in a degassed DMF/carbonate buffer solvent mixture. The successful attachment of the boronic acid was evidenced by HPLC separation from phenol side products and clear identification via MALDI-TOF mass spectrometry as a citric acid derivative. This postsynthetic chemistry was further combined with the established Cu(I)-catalyzed azide-alkyne cycloaddition chemistry to allow the first orthogonal and postsynthetic incorporation of both the phenylboronic acid moiety and two different cyanine-styryl dyes. Because of the undesired reactivity of boronic acids by the presence of copper salts, the dye azides were first attached to the presynthesized oligonucleotides using the Cu(I)-catalyzed cycloaddition at the 2'-position of a propargylated uridine. After careful removal of all copper contaminants, the amide bond with the 4-carboxyphenylboronic acid at the propylamine linker of a 7-deaza-2'-deoxyadenosine as anchor point was formed. These doubly modified oligonucleotides were characterized by their optical properties to elucidate the influence of the phenylboronic acid. The latter modification has only little influence on the fluorescence of the applied dyes. In conclusion, this postsynthetic and orthogonal chemistry opens the way to a broad variety of applications, in particular, saccharide detection based on fluorescent DNA aptamers.

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

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.

"DNA Origami Traffic Lights" with a Split Aptamer Sensor for a Bicolor Fluorescence Readout.

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.

Polarity Sensitive Bioorthogonally Applicable Far-Red Emitting Labels for Postsynthetic Nucleic Acid Labeling by Copper-Catalyzed and Copper-Free Cycloaddition.

Two series of new, water-soluble, membrane-permeable, far-red/NIR emitting benzothiazolium-based fluorescent labels with large Stokes' shifts were synthesized that can be conjugated to alkyne-modified biomolecules through their azide moiety via azide-alkyne cycloaddition. We have used these azide bearing labels to make fluorescent DNA constructs using copper-catalyzed "click" reaction. All dyes showed good or remarkable fluorescence intensity enhancement upon conjugation to DNA. We also investigated the possibility to incorporate the benzocyclooctyne motif through rigid (ethnynyl) or flexible (ethyl) linkers into the DNA, thus enabling copper-free labeling schemes. We observed that there is a marked difference between the two linkers applied in terms of optical properties of the labeled oligonucleotides. We have also tested the in vivo labeling potential of these newly synthesized dyes on HeLa cells previously transfected with cyclooctynylated DNA. Confocal fluorescent images showed that the dyes are all able to cross the membrane and suitable for background-fluorescence free fluorescent tagging of nucleic acids. Moreover, we have observed different accumulation of the two dye series in the endosomal particles, or in the nuclei, respectively.

Synthesis of Wavelength-Shifting Fluorescent DNA and RNA with Two Photostable Cyanine-Styryl Dyes as the Base Surrogate Pair.

Two nucleic acid building blocks were synthesized, consisting of two photostable green- and red-emitting cyanine-styryl dyes and (S)-3-amino-1,2-propanediol as a substitute for the ribofuranoside, and incorporated as base-pair surrogates by using automated phosphoramidte chemistry in the solid phase. The optical properties and, in particular, the energy-transfer properties were screened in a range of DNA duplexes, in which the "counter bases" of the two dyes were varied and the distance between the two dyes was enlarged to up to three intervening adenosine-thymidine pairs. The DNA duplex with the best optical properties and the best red/green emission ratio as the readout bore adenosine and thymidine opposite to the dyes, and the two dyes directly adjacent to each other as the base surrogate pair. This structural arrangement can be transferred to RNA to obtain similarly fluorescent RNA probes. Representatively, the positively evaluated DNA duplex was applied to verify the fluorescence readout in living HeLa cells by using fluorescence confocal microscopy.