An electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targets.

The specific detection in clinical samples of analytes with dimensions in the tens to hundreds of nanometers, such as viruses and large proteins, would improve disease diagnosis. Detection of these "mesoscale" analytes (as opposed to their nanoscale components), however, is challenging as it requires the simultaneous binding of multiple recognition sites often spaced over tens of nanometers. In response, we have adapted DNA origami, with its unparalleled customizability to precisely display multiple target-binding sites over the relevant length scale, to an electrochemical biosensor platform. Our proof-of-concept employs triangular origami covalently attached to a gold electrode and functionalized with redox reporters. Electrochemical interrogation of this platform successfully monitors mesoscale, target-binding-induced changes in electron transfer in a manner consistent with coarse-grained molecular dynamics simulations. Our approach enables the specific detection of analytes displaying recognition sites that are separated by ∼40 nm, a spacing significantly greater than that achieved in similar sensor architectures employing either antibodies or aptamers.

Designing Higher Resolution Self-Assembled 3D DNA Crystals via Strand Terminus Modifications.

DNA tensegrity triangles self-assemble into rhombohedral three-dimensional crystals via sticky ended cohesion. Crystals containing two-nucleotide (nt) sticky ends (GA:TC) have been reported previously, and those crystals diffracted to 4.9 Å at beamline NSLS-I-X25. Here, we analyze the effect of varying sticky end lengths and sequences as well as the impact of 5'- and 3'-phosphates on crystal formation and resolution. Tensegrity triangle motifs having 1-, 2-, 3-, and 4-nt sticky ends all form crystals. X-ray diffraction data from the same beamline reveal that the crystal resolution for a 1-nt sticky end (G:C) and a 3-nt sticky end (GAT:ATC) were 3.4 and 4.2 Å, respectively. Resolutions were determined from complete data sets in each case. We also conducted trials that examined every possible combination of 1-nucleotide and 2-nucleotide sticky-ended phosphorylated strands and successfully crystallized all 16 possible combinations of strands. We observed the position of the 5'-phosphate on either the crossover (1), helical (2), or central strand (3) affected the resolution of the self-assembled crystals for the 2-turn monomer (3.0 Å for 1-2P-3P) and 2-turn dimer sticky ended (4.1 Å for 1-2-3P) systems. We have also examined the impact of the identity of the base flanking the sticky ends as well as the use of 3'-phosphate. We conclude that crystal resolution is not a simple consequence of the thermodynamics of the direct nucleotide pairing interactions involved in molecular cohesion in this system.

Crystal structure of a DNA/Ba2+ G-quadruplex containing a water-mediated C-tetrad.

We have determined the 1.50 Å crystal structure of the DNA decamer, d(CCA(CNV)KGCGTGG) ((CNV)K, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba(2+). The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba(2+) ions to occupy adjacent steps in the central ion channel. One ordered Mg(2+) facilitates 3'-3' stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the (CNV)K nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson-Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation.

Photocleavage control of nucleated DNA nanosystems--the influence of surface strand sterics.

We use sterically inaccessible 'seed' strands, released from a surface into solution by photocleavage to initiate a nucleated DNA polymerization reaction. We demonstrate control of the quantity of 'seed' release and that hairpin steric protection of the 'seed' leads to less 'leaky' surfaces. This polymerization is a model system for surface-photocleavage initiation of sub-stoichiometric reaction cascades; these cascades should find use as a component of labs-on-chips capable of bioanalytical and DNA-computing tasks.

Hetero-oligonucleotide nanoscale tiles capable of two-dimensional lattice formation as testbeds for a rapid, affordable purification methodology.

New nanoscale hetero-oligonucleotide tiles are assembled from DNA, RNA and morpholino oligos and purified using size exclusion filtration. Homo-oligonucleotide tiles assembled from RP-cartridge processed DNA oligos are purified by nondenaturing gel electrophoresis. These tiles' purity and homogeneity are demonstrated by gel electrophoresis and their incorporation into two-dimensional arrays visualized by AFM. This purification methodology increases throughput and decreases costs for researchers who wish to screen multiple tiles for utilization in structural or analytical studies.

Positional photocleavage control of DNA-based nanoswitches.

DNA-based switches are currently operated by manual solution-phase addition of 'set-strands'. We demonstrate another operation method-sterically inaccessible 'set-strands', released from a surface into solution by spatially controlled photocleavage. This technique will enable microarrays of set-strands to operate many DNA-based switches in self-contained computational and diagnostic devices.

Metallic nanoparticles used to estimate the structural integrity of DNA motifs.

Branched DNA motifs can be designed to assume a variety of shapes and structures. These structures can be characterized by numerous solution techniques; the structures also can be inferred from atomic force microscopy of two-dimensional periodic arrays that the motifs form via cohesive interactions. Examples of these motifs are the DNA parallelogram, the bulged-junction DNA triangle, and the three-dimensional-double crossover (3D-DX) DNA triangle. The ability of these motifs to withstand stresses without changing geometrical structure is clearly of interest if the motif is to be used in nanomechanical devices or to organize other large chemical species. Metallic nanoparticles can be attached to DNA motifs, and the arrangement of these particles can be established by transmission electron microscopy. We have attached 5 nm or 10 nm gold nanoparticles to every vertex of DNA parallelograms, to two or three vertices of 3D-DX DNA triangle motifs, and to every vertex of bulged-junction DNA triangles. We demonstrate by transmission electron microscopy that the DNA parallelogram motif and the bulged-junction DNA triangle are deformed by the presence of the gold nanoparticles, whereas the structure of the 3D-DX DNA triangle motif appears to be minimally distorted. This method provides a way to estimate the robustness and potential utility of the many new DNA motifs that are becoming available.

Thermodynamic analysis of nylon nucleic acids.

The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides that contained nylon nucleic acids (1-5 amide linkages) revealed that the amide linkage significantly enhanced the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26 degrees C increase in the thermal transition temperature (T(m)) for five linkages) and RNA (around 15 degrees C increase in T(m) for five linkages) compared with nonamide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised T(m) from less to slightly greater than T(m) of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2'-position of ribose and recovery of stability upon linkage of the side chains.

Morphology Change of Calcium Carbonate in the Presence of Two-Dimensional DNA Lattices.

Two-dimensional DNA lattices are grown under conditions that also are suitable for the magnesium-free growth of three-dimensional calcium carbonate crystals. These lattices are used to template morphology changes in calcium carbonate. The effects of DNA lattices, sub-assemblies, duplexes, single strands, dinucleotides, and mononucleotides on calcium carbonate morphology are studied. A "rotated" morphology of calcite is found to predominate when a critical concentration of any polynucleotide is reached in the templating solution.

Experiments in Structural DNA Nanotechnology: Arrays and Devices.

In recent years, the chemistry of DNA has expanded from biological systems to nanotechnology. The generalization of the biological processes of reciprocal exchange leads to stable branched motifs that can be used for the construction of DNA-based geometrical and topological objects, arrays and nanomechanical devices. The information in DNA is the basis of life, but it can also be used to control the physical states of a variety of systems, leading ultimately to nanorobotics; these devices include shape-changing, walking and translating machines. We expect ultimately to be able to use the dynamic information-based architectural properties of nucleic acids to be the basis for advanced materials with applications from nanoelectronics to biomedical devices on the nanometer scale.

Nucleic Acid Nanostructures: Bottom-Up Control of Geometry on the Nanoscale.

DNA may seem an unlikely molecule from which to build nanostructures, but this is not correct. The specificity of interaction that enables DNA to function so successfully as genetic material also enables its use as a smart molecule for construction on the nanoscale. The key to using DNA for this purpose is the design of stable branched molecules, which expand its ability to interact specifically with other nucleic acid molecules. The same interactions used by genetic engineers can be used to make cohesive interactions with other DNA molecules that lead to a variety of new species. Branched DNA molecules are easy to design, and the can assume a variety of structural motifs. These can be used for purposes both of specific construction, such as polyhedra, and for the assembly of topological targets. A variety of two-dimensional periodic arrays with specific patterns have been made. DNA nanomechanical devices have been built with a series of different triggers, small molecules, nucleic acid molecules and proteins. Recently, progress has been made in self-replication of DNA nano-constructs, and in the scaffolding of other species into DNA arrangements.

Two dimensional PNA/DNA arrays: estimating the helicity of unusual nucleic acid polymers.

We extend the generality of nucleic acid-based structural nanotechnology by incorporating non-natural nucleic acids into a DNA double crossover (DX) molecule; visualizing two-dimensional arrays of these DX molecules by Atomic Force Microscopy (AFM) enables us to measure the helical repeat of any heteroduplex sequence capable of forming the outer arms of a DX.

Nylon/DNA: Single-stranded DNA with a covalently stitched nylon lining.

The synthesis of DNA/nylon ladder oligomers is described. Three stages of the development are addressed: the synthesis of 2'-beta-substituted phosphoramidites, the deprotection/purification protocols of ODNs modified with both amino and carboxyl groups, and amide bond-forming reactions on the ODNs. The established technology and the novel DNA-based ladder oligomer structure opens a pathway to the synthesis of topological molecular objects and networks templated by DNA through versatile DNA nanotechnology. The DNA-based ladder oligomers may find application in the antisense area.