Yoel P. Ohayon

Functionalizing Designer DNA Crystals with a Triple-Helical Veneer†

DNA is a very useful molecule for the programmed self-assembly of 2D and 3D nanoscale objects.[1] The design of these structures exploits Watson–Crick hybridization and strand exchange to stitch linear duplexes into finite assemblies.[2–4] The dimensions of these complexes can be increased by over five orders of magnitude through self-assembly of cohesive single-stranded segments (sticky ends).[5,6] Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,[7] the direction of biological cascades,[8] and the structure determination of periodically positioned molecules by X-ray diffraction.[9] To this end we present a macroscopic 3D crystal based on the 3-fold rotationally symmetric tensegrity triangle[3,6] that can be functionalized by a triplex-forming oligonucleotide on each of its helical edges.

Topological Linkage of DNA Tiles Bonded by Paranemic Cohesion

Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.

Construction and Structure Determination of a Three-Dimensional DNA Crystal

Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.

Self-Assembly of 3D DNA Crystals Containing a Torsionally Stressed Component

There is an increasing appreciation for structural diversity of DNA that is of interest to both DNA nanotechnology and basic biology. Here, we have explored how DNA responds to torsional stress by building on a previously reported two-turn DNA tensegrity triangle and demonstrating that we could introduce an extra nucleotide pair (np) into the original sequence without affecting assembly and crystallization. The extra np imposes a significant torsional stress, which is accommodated by global changes throughout the B-DNA duplex and the DNA lattice. The work reveals a near-atomic structure of naked DNA under a torsional stress of approximately 14%, and thus provides an example of DNA distortions that occur without a requirement for either an external energy source or the free energy available from protein or drug binding.

132 Impact of sticky end length on the diffraction of self-assembled DNA crystals

Our laboratory has reported a self-assembled 3-D crystal based on a DNA tensegrity triangle. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry consisting of three helices whose axes are directed along three linearly independent directions (1). The triangles form a crystalline lattice stabilized via sticky ends (2). The length of the sticky ends reported previously was two nucleotides (nt) GA:TC. Although diffracting to 4 Å resolution at the APS-ID19 beam line, they diffract only to 4.9 Å at the NSLS-X25 beam line. In the current study, we have analysed the effect of sticky end length and sequence on crystal formation and the resolution of the X-ray diffraction pattern on NSLS-X25. Tensegrity triangle motifs having 1-, 2-, and 3-nt sticky ends have all formed crystals. X-ray diffraction data from the same beam line revealed that the crystal resolution was somewhat better for the 2-nt sticky end having an AA:TT base pair (4.75 Å) than GA:CT and CC:GG (8.0 Å). Moreover, the 1-nt sticky end (C:G) yielded a diffraction pattern whose resolution (3.5 Å) compared favorably with all the three 2-nt sticky end systems. However, the triangle motif having a 1-nt sticky end with an A:T base pair did not yield any crystals. For motifs with 3-nt sticky ends, the sequence GAG:CTC produced small crystals (10–20 μm), while larger crystals (150 μm) were obtained with the sequences TAG:ATC and TAT:ATA. Our results indicate that not only do the lengths and sequences of the sticky ends define the interactions between motifs, but they also have an impact on the resulting resolution. We expect redesigned assemblies to form 3-D crystals with better resolution that can aid in the scaffolding of biological macromolecules for crystallographic structure determination. Applications in many areas of DNA nanotechnology are expected to benefit from a complete analysis of the effects of sticky end length, sequence, and free energy.

78 Programmable crystal contacts used to improve the resolution of self-assembled 3D DNA crystals

Gu, H., Chao, J., Xiao, S. J., & Seeman, N. C. (2010). A proximity-based programmable DNA nanoscale assembly line. Nature, 465, 202–205. Seeman, N. C. (2005). From genes to machines: DNA nanomechanical devices. Trends in Biochemical Sciences, 30, 119–125. Zheng, J., Birktoft, J. J., Chen, Y., Wang, T., Sha, R., Constantinou, P. E., ... Seeman, N. C. (2009). From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature, 461, 74–77.

77 Let’s get twisted: the crystal structure of torsionally stressed DNA

Gu, H., Chao, J., Xiao, S. J., & Seeman, N. C. (2010). A proximity-based programmable DNA nanoscale assembly line. Nature, 465, 202–205. Seeman, N. C. (2005). From genes to machines: DNA nanomechanical devices. Trends in Biochemical Sciences, 30, 119–125. Zheng, J., Birktoft, J. J., Chen, Y., Wang, T., Sha, R., Constantinou, P. E., ... Seeman, N. C. (2009). From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature, 461, 74–77.

133 Designed DNA crystals with a triple-helix veneer

DNA has been used as a tool for the self-assembly of nano-sized objects and arrays in two and three-dimensions. Triplex-forming oligonucleotides (TFOs) can be exploited to recognize and introduce functionality at precise duplex regions within these DNA nanostructures (Rusling et al., 2012). Here we have examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry, consisting of three helices directed along three linearly independent directions (Liu et al., 2004). The triangles form a three-dimensional crystalline lattice stabilized via sticky-end cohesion (Zheng et al., 2009). The TFO 5′-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine–oligopyrimidine binding site. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET analysis. Non-denaturing gel analysis of annealed DNA motifs showed a band with slower mobility only in the presence of TFO and only when the DNA motif contained the triplex binding site. Experiments were undertaken at pH 5.0, since the formation of a triplex with cytidine-containing TFOs requires slightly acidic conditions (pH< 6.0). TFOs with modified C-analogs and T-analogs having a higher pK a worked at a more neutral pH, also evidenced by EMSA. UV melting studies revealed that the melting point of the 3-turn triangle was 64 °C and the TFO binding increased the melting point to 80 °C. FRET analysis was done by labeling the triangle with fluorescein and the TFO with a cyanine dye (Cy5). The FRET melting curve revealed that a signal was observed only when the TFO was bound to the DNA motif and the results were consistent with UV melting studies. These results indicate that a TFO can be specifically targeted to the tensegrity triangle motif.

Paranemic Crossover DNA: There and Back Again

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX2, has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.