1. Fig. 3. AFM images of (A) 1D cross origami produced using the traditional 18- hour anneal, (B) 2D cross origami produced using the traditional 18-hour anneal, (C) 1D cross origami produced using the rapid 30-minute anneal, and (D) 2D cross origami produced using the rapid 30-minute anneal. A. B. C.D. Fig. 3 A and B show 1D and 2D cross origami, respectively. These origami were annealed using the eighteen-hour method in the real-time thermal controller. The 1D origami, though tightly clustered, was well formed (The “equal signs” discussed in Fig. 1 are present.), and the chains had typical lengths of ~1µm, as we have seen in previous experiments. The 2D origami also appeared to have formed adequately and were arranged in fairly large arrays. Fig. 3 C and D, too, show 1D and 2D cross origami, respectively. This time, though, the origami were annealed using the thirty-minute thermal-cycler method instead. The 1D origami, like that of the eighteen- hour run, had precise form (“equal signs”). However, the chains appeared to be longer (> 1µm) than those featured in Fig. 4 A. (Considering that each cross is only about 100 nm in width, a 1.7-micrometer chain is huge!) The 2D origami was especially pleasing, featuring what looked to be a very solid 6x6 cross array (“equal signs”). In only thirty minutes, we were able to produce origami that was qualitatively very similar to origami produced over an eighteen-hour period. This allows for quick turnaround during future experiments which is necessary when future plans depend upon the answers that may lie within a present experiment. The results of this experiment, and the prospect of a quick alternative to a conventional method, are encouraging. 1) Rothemund, Paul W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature. 2006, 440, 297-302. 2) Liu, Wenyan; Zhong, Hong; Wang, Risheng; Seeman, Nadrian C. Crystalline Two-Dimensional DNA-Origami Arrays. Angewandte Chemie International Edition. 2011, 50, 264-267. 3) Arbona, Jean-Michel; Elezgaray, Juan; Aime, Jean-Pierre. Modelling the Folding of DNA Origami. Europhysics Letters. 2012, 100, 28006. Funding for this project was provided by the U.S. Army through the High School Apprenticeship Program (HSAP). Tanner Bakhshi would like to thank Dr. Michael Norton, David Neff, and Dr. Masudur Rahman for guidance and support. Tanner Bakhshi, Molly McIlvain, David Neff, and Michael Norton, Ph.D.: Department of Chemistry, Marshall University. In 2006 Paul W. K. Rothemund of Caltech published an article in Nature about something he called “DNA Origami,” 1 a technique involving the manipulation or folding of DNA to form virtually any desired shape or pattern. In the seven years following this discovery, DNA Origami has become a sensational research topic in many disciplines, namely the field of DNA nanotechnology. With the many potential benefits of this technology, however, come many challenges. One challenge in particular is the length of time that it can take to anneal a long DNA strand to short “staple strands” that “show” the longer strand of DNA how and where to fold. Standard protocol for this procedure calls for an eighteen-hour temperature ramp using a Polymerase Chain Reaction (PCR) machine. By staining the DNA with a fluorescent nucleic acid stain (SYBR Green I, Cambrex), it is possible to obtain fluorescent intensity data while annealing DNA origami. As more and larger DNA structures begin to form, fluorescence increases proportionally, thus providing a map that highlights temperatures that are crucial in origami formation. After analyzing this data, it was evident that multiple temperature ranges were not instrumental in the formation of origami. It was hypothesized that if a temperature ramp protocol including only those ranges that appeared to be critically important in origami formation were developed, then origami could be annealed in a significantly shorter amount of time, a mere thirty minutes. Results showed that the thirty-minute protocol was capable of forming origami after only this short annealing time. Using atomic force microscopy (AFM), samples of origami from both protocols were characterized and compared. The samples were determined to be equally sound in form and structure. The shorter, thirty-minute method of origami formation can be used as a viable alternative to the eighteen-hour procedure when fast experimental turnaround is desired. In 2006 Paul W. K. Rothemund, a researcher at the California Institute of Technology, published an article in Nature about something he called “DNA Origami.” 1 True to the way that it sounds, DNA Origami involves the folding of DNA into different patterns and shapes through the use of “staple strands” that tell the DNA where and how to fold. The type of origami used in this experiment is called “cross” origami. 2 Just like it sounds, the shape is formed by the joining of two linked but individually- formed rectangular DNA structures, the end result’s being what can be described as a cross or plus sign. Depending on how the ends of these separate structures are designed, the crosses formed can be individual, they can stick together in one direction and form long chains, or they can stick together in two directions and form large checkerboard-like arrays. These different configurations are referred to as 0D, 1D, and 2D crosses, respectively. Though DNA Origami has become a sensational research topic in many disciplines and poses many potential benefits, studying it does not come without challenges. One challenge in particular is the length of time that it can take to anneal a long DNA strand to the aforementioned staple strands. Standard protocol for this procedure calls for an eighteen- hour temperature ramp. It was hypothesized that if a temperature ramp protocol including only those ranges that appeared to be critically important in origami formation were developed, then origami could be annealed in a significantly shorter amount of time, a mere thirty minutes. Fig. 2. Graphs showing (A) the derivatives (ΔF/ΔT) taken of the numerical values of intensity at each temperature during 1D cross origami formation, (B) the amount of time spent at each temperature in the thirty-minute program for 1D cross origami, (C) the derivatives (ΔF/ΔT) taken of the numerical values of intensity at each temperature during 2D cross origami formation, and (D) the amount of time spent at each temperature in the thirty-minute program for 2D cross origami formation. Note that annealing programs (B and D) were made after derivative data was gathered and analyzed (A and C). A. B. C. D.