Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
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 pKa 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.
We acknowledge support of the following grants to NCS: grant GM-29554 from NIGMS, grants CTS-0608889 and CCF-0726378 from the NSF, grant W911FF-08-C-0057 from ARO, grants N000140910181 and N000140911118 from ONR and DE-SC0007991 from DOE.
Rusling, D. A., Nandhakumar, I. S., Brown, T. and Fox K. R. (2012). Triplex-directed recognition of a DNA nanostructure assembled by crossover strand exchange. ACS Nano 6, 3604-3613.
Zheng, J., Birktoft, J. J., Chen, Y., Wang, T., Sha, R., Constantinou, P. E., Ginell, S. L., Mao, C and Seeman, N. C. (2009). From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature 461, 74-77.
Arun Richard Chandrasekaran
Department of Chemistry