Origami is the art of paper folding that has been practiced in Japan since Edo period (1603-1868), and then successfully introduced in the western culture. The goal of this art is to transform a dull piece of paper into sophisticated 3D sculptures by the sequence of folding and sculpting. Likewise Japanese origami, DNA origami is also a way of 3D-structures folding – folding in the nanoscale. In this technique instead of sheet of paper one uses hundreds of short DNA strands to fold a long circular DNA molecule (scaffold) into 3D DNA-based structures. In this post, I will try to convince you that DNA origami technique is nothing to be scared about. Frankly speaking, once you gain some basic knowledge and skills, it might be even easier to fold your fancy sculpture with DNA than using paper (at least if you have enough money and lab-facility to work with 🙂 ).
The first step in the synthesis of your own DNA origami is to design a structure that will live up to your expectations. DNA origami can have many functionalities that strongly rely on their 3D-architecture. Therefore, firstly you have to think about the morphology of your DNA origami and how to connect adjacent DNA bundles to fold them in an exact way. It might sounds easy, but as always, the devil is in the detail. If you wanted to make a design by hand, you would have to find a way to fold the circular DNA strand containing few thousand nucleotides into a 3D structure using hundreds of short DNA staples. Furthermore, you would have to remember about DNA base pair complementarity and its geometry, and inset crossovers to make the structure more rigid and stable. It is fair to say that designing DNA origami “by hand” is not an easy task at all, nevertheless, it has been done this way in the early days. Thankfully, we today can benefit from some software helping us with designing, where caDNAno (www.cadnano.org) is the most popular one. In caDNAno the user interface is divided into three main panels (see Fig. 1). Left panel represent a cross-section of your DNA origami on which you can define number of DNA bundles. In the middle panel, you can determine the length of each bundle, while the right one displays the 3D model of your DNA origami design. Moreover, the software support you with the information about short DNA strands that are required to fold your structure.
Once you have your scaffold and DNA staples you can start folding your design. Typical assembly of DNA origami is carried out in a one-pot reaction with the specific buffer and a source of magnesium ions. Buffer, typically Tris-EDTA or Tris-acetate-EDTA, secures stable folding conditions, while magnesium ions shield negative charge of DNA enabling it to form dense 3D structure. To ensure high yield and accuracy of the reaction, the whole process is carried out in a thermocycler that provides stable thermal cycles (see Fig. 2). Briefly, folding solution is annealed in high temperature and then slowly cools down to ensure accurate assembly of DNA strands into DNA origami.
Once the reaction is finished, you have your own DNA origami in a test tube. Well, it is not really your DNA origami, or at least not only. After the folding reaction is finished, you end up with a mixture of DNA origami, excess DNA strands, and some amount of aggregates. In order to get rid of aggregates and DNA staples we conduct gel electrophoresis. Briefly, samples are loaded into wells of an agarose gel and then subjected to an electric field causing the separation of different DNA structures (see Fig. 3). Afterwards, the gel is stained with DNA-intercalating dye, which under UV light irradiation starts to fluoresce enabling us to cut out the band containing DNA origami and collect the solution into the test tube.
At this stage, we hold the test tube with purified DNA origami, but obviously, we would like to see something more than the transparent solution after hours and hours of hard work. As DNA origami structures are extremely small (millions times smaller than paper origami sculptures) we definitely cannot see them by the naked eye. To be able to visualize your minuscule DNA origami, you have to use something much more powerful – an electron microscope. The electron microscope, instead of light, uses beam of extremely accelerated electrons to reveal the structure of studied objects. An example the electron microscope image of DNA origami that I used in my studies is presented in Figure 4.
So there you have it! This was my very brief guideline about the synthesis of DNA origami. I believe that now you will feel much more competent to immerse into the realm of DNA nanotechnology – and trust me there is still a lot to explore.
This online Lecture was written by Karol Kolataj from the Liedl Group at LMU Munich