LETTERS 38 nature materials | VOL 3 | JANUARY 2004 | www.nature.com/naturematerials D NA possesses many desirable chemical/physical properties as a polymeric material. With the myriad of tools available to manipulate DNA 1 , there is great potential for using DNA as a generic instead of a genetic material.Although much progress has been made in DNA computing 2–4 and DNA nanotechnology 5–19 , the full achievement of DNA-based materials has not yet been realized. As almost all DNA molecules are either linear or circular, to rationally construct DNA materials one must first create additional shapes of DNA as basic building blocks. In addition, these DNA building blocks must be readily incorporated into larger structures in a controlled manner. Here, we show the controlled assembly of dendrimer-like DNA (DL-DNA) from Y-shaped DNA (Y-DNA). The synthesis of Y-DNA and controlled assembly of DL-DNA were robust and efficient; the resulting DL-DNA was stable and almost monodisperse. The multivalent DNA dendrimers can be either isotropic or anisotropic, providing great potential to link other entities. Two strategies were used to synthesize the Y-DNA: stepwise synthesis and all-in-one (one-pot) synthesis (Fig. 1a). In the stepwise approach, two oligonucleotides with partial complementary sequences formed one arm of a Y-DNA; then the third oligonucleotide that was complementary to the first two unmatched portions of oligonucleotides, formed the other two arms of the Y-DNA. In the one- pot approach, equal moles of all three oligonucleotides were mixed together to form the Y-DNA.In both cases,the formation of Y-DNA was evaluated by gel electrophoresis (Fig. 1c), in which the mobility of a DNA molecule depends on its size, shape and extent of base pairing 20 . One major band appears on the gel (Fig. 1c, lanes 4–6), and its mobility is less than that of its components, the single-stranded DNA (lanes 1–3), indicating the formation of one arm of Y-DNA. The further shift of the mobility of the final annealing product of stepwise (Fig. 1c, lanes 7–9) and one-pot synthesis (Fig. 1c, lane 10) indicated the formation of Y-DNA. There is no difference in results between stepwise and one-pot synthesis.The estimated yield of Y-DNA is close to 100%,as estimated by densitometry. Other Y-DNA with different sequences were similarly synthesized. Synthesized Y-DNA were stable with no degradation observed after 30 days at 4 °C (Fig. 1d). DL-DNA were assembled by ligation of Y-DNA molecules, whose sequences were specifically designed so that ligations between Y i and Y j DNA could only occur when i ≠ j, where i and j refer to the generation number n (for example, G 1 ,G 2 , etc., see Fig. 3a. The cohesive end of each oligonucleotide was non-palindromic, thus no self-ligations occurred, see Table 1, segment 1). In addition, the ligation could only occur in one direction, that is,Y 0 →Y 1 →Y 2 →Y 3 →Y 4 and so on. Furthermore, when Y 0 was ligated to Y 1 with a 1:3 molar stoichiometry, one Y 0 was linked with three Y 1 ,forming the first-generation DL-DNA (G 1 ,Fig. 2a).G 1 was then ligated to six Y 2 (one Y 2 for each of the six free branches of G 1 ), resulting in a second-generation DL-DNA (G 2 , Fig. 3a). The third (G 3 ), fourth (G 4 ), and higher generation DL-DNA were assembled in a similar way (Fig. 3a). Note that the assembled DL-DNA (G n ) had only one possible conformation due to the unidirectional ligation strategy. The general format of the n th -generation DL-DNA is G n = (Y 0 )(3Y 1 )(6Y 2 )…(3 × 2 n–1 Y n ), where n is the generation number and Y n is the n th Y-DNA. The total number of Y-DNA in an n th -generation DL-DNA is 3 × 2 n – 2. The growth of DL-DNA from n th generation to (n + 1) th generation requires a total of 3 × 2 n new Y n+1 -DNA. G 1 DL-DNA were assembled by ligating Y 0 and Y 1 with a 1:3 stoichiometry (Fig. 2a). The ligation product migrated as a single band, and its mobility was slower than that of its building block, Y 0 (Fig. 2c). The presence of a single band indicated a new molecular species with a well-defined molecular weight. The estimated yield is close to 100%. To confirm that the ligation product was indeed G 1 DL-DNA, it was denatured (Fig. 2b) and examined by gel electrophoresis (Fig. 2d). Two major bands appeared in the electrophoresis: one with the same mobility as the single-strand DNA Y 0a (30-mer) and one with slower mobility (90-mer), which was exactly what one would expect from the G 1 DL-DNA structure according to the assembly scheme (Fig. 2b). Similar results were obtained from denaturation of G 2 ,G 3 and G 4 , and the generation of newly ligated species were revealed by electrophoresis (data not shown). Assembled G 1 DL-DNA were stable with no degradation observed after 45 days at 4 °C (Fig. 2e). In addition, exonuclease III assays confirmed the absence of cyclic materials (data not shown). The second-, third-, fourth- and fifth-generation DL-DNA were synthesized with a similar strategy and evaluated by gel electrophoresis (Fig. 3a and 3b). With increasing generation, the mobility of the ligated product decreased as predicted (Fig. 3b, see arrows). Furthermore, the yield and the purity of higher generation DL-DNA did not seem to decrease even in the absence of purification, suggesting that the assembly was very robust. To further confirm that the mobility-shifted species were indeed DL-DNA molecules, we examined the 4 th generation DL-DNA by atomic force microscopy (AFM) with both a Controlled assembly of dendrimer-like DNA YOUGEN LI 1 , YOLANDA D. TSENG 1 , SANG Y. KWON 1 , LEO D’ESPAUX 1 , J. SCOTT BUNCH 2 , PAUL L. MCEUEN 2 AND DAN LUO* 1 1 Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853-5701, USA 2 Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853-5701, USA *e-mail: DL79@cornell.edu Published online: 21 December 2003; doi:10.1038/nmat1045 ©2004 Nature Publishing Group