Asymmetric patterning drives the folding of a tripodal DNA nanotweezer.

DNA tweezers have emerged as powerful devices for a wide range of biochemical and sensing applications; however, most DNA tweezers consist of single units activated by DNA recognition, limiting their range of motion and ability to respond to complex stimuli. Herein, we present an extended, tripodal DNA nanotweezer with a small molecule junction. Simultaneous, asymmetric elongation of our molecular core is achieved using polymerase chain reaction (PCR) to produce length- and sequence-specific DNA arms with repeating DNA regions. When rigidified, our DNA tweezer can be addressed with streptavidin-binding ligands. Full control over the number, separation, and location of these ligands enables site-specific streptavidin recognition; all three arms of the DNA nanotweezer wrap around multiple streptavidin units simultaneously. Our approach combines the simplicity of DNA tile arrays with the size regime normally provided by DNA origami, offering an integrated platform for the use of branched DNA scaffolds as structural building blocks, protein sensors, and dynamic, stimuli-responsive materials.

Selection of a metal ligand modified DNAzyme for detecting Ni2.

Nickel is a highly important metal, and the detection of Ni2+ using biosensors is a long-stand analytical challenge. DNA has been widely used for metal detection, although no DNA-based sensors were reported for Ni2+. DNAzymes are DNA-based catalysts, and they recruit metal ions for catalysis. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 50 random nucleotides in the presence of Ni2+. To increase Ni2+ binding, a glycyl-histidine-functionalized tertiary amine moiety was inserted at the cleavage junction. A representative DNAzyme named Ni03 showed a high cleavage yield with Ni2+ and it was further studied. After truncation, the optimal sequence of Ni03l could bind one Ni2+ or two Co2+ for catalysis, while other metal ions were inactive. Its cleavage rates for 100 µM Ni2+ reached 0.63 h-1 at pH 8.0. A catalytic beacon biosensor was designed by labeling a fluorophore and a quencher on the Ni03l DNAzyme. Fluorescence enhancement was observed in the presence of Ni2+ with a detection limit of 12.9 µM. The sensor was also tested in spiked Lake Ontario water achieving a similar sensitivity. This is another example of using single-site modified DNAzyme for sensing transition metal ions.

Target Self-Enhanced Selectivity in Metal-Specific DNAzymes.

Highly selective recognition of metal ions by rational ligand design is challenging, and simple metal binding by biological ligands is often obscured by nonspecific interactions. In this work, binding-triggered catalysis is used and metal selectivity is greatly increased by increasing the number of metal ions involved, as exemplified in a series of in vitro selected RNA-cleaving DNAzymes. The cleavage junction is modified with a glycyl-histidine-functionalized tertiary amine moiety to provide multiple potential metal coordination sites. DNAzymes that bind 1, 2, and 3 Zn2+ ions, increased their selectivity for Zn2+ over Co2+ ions from approximately 20-, 1000-, to 5000-fold, respectively. This study offers important insights into metal recognition by combining rational ligand design and combinatorial selection, and it provides a set of new DNAzymes with excellent selectivity for Zn2+ ions.

Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale.

Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA compounds. Nuclease degradation of the strands outside and inside the cell results in a misleading intracellular fluorescent signal. The signal is saturated by the fluorescence of the degradation product (phosphorylated dye). To test our hypothesis, we synthesized a range of DNA structures, including Cy3- and Cy5-labeled DNA cubes and DNA tetrahedra, and oligonucleotides with different stabilities toward nucleases. All give fluorescence signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control. Kinetics experiments revealed that uptake of stable DNA structures is delayed. We also studied several parameters influencing fluorescent data: stability of the DNA strand, fixation methods that can wash away the signal, position of the dye on the DNA strand, and design of FRET experiments. DNA nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility, programmability, and easy synthesis. However, few examples of successful DNA machines in vivo have been reported. We believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent dyes, in order to further propel the biological development, and application of DNA nanostructures.

"Printing" DNA Strand Patterns on Small Molecules with Control of Valency, Directionality, and Sequence.

The incorporation of synthetic molecules as corner units in DNA structures has been of interest over the last two decades. In this work, we present a facile method for generating branched small molecule-DNA hybrids with controllable valency, different sequences, and directionalities (5'-3') using a "printing" process from a simple 3-way junction structure. We also show that the DNA-imprinted small molecule can be extended asymmetrically using polymerase chain reaction (PCR) and can be replicated chemically. This strategy provides opportunities to achieve new structural motifs in DNA nanotechnology and introduce new functionalities to DNA nanostructures.

Modular Strategy To Expand the Chemical Diversity of DNA and Sequence-Controlled Polymers.

Sequence-defined polymers with customizable sequences, monodispersity, substantial length, and large chemical diversity are of great interest to mimic the efficiency and selectivity of biopolymers. We report an efficient, facile, and scalable synthetic route to introduce many chemical functionalities, such as amino acids and sugars in nucleic acids and sequence-controlled oligophosphodiesters. Through achiral tertiary amine molecules that are perfectly compatible with automated DNA synthesis, readily available amines or azides can be turned into phosphoramidites in two steps only. Individual attachment yields on nucleic acids and artificial oligophosphodiesters using automated solid-phase synthesis (SPS) were >90% in almost all cases. Using this method, multiple water-soluble sequence-defined oligomers bearing a range of functional groups in precise sequences could be synthesized and purified in high yields. The method described herein significantly expands the library of available functionalities for nucleic acids and sequence-controlled polymers.