Hello there! It’s Néstor Sampedro writing again from Aarhus, Denmark. I am part of the Andersen lab in which we are designing novel RNA molecules for biotechnological applications. Nanostructures made out of this molecule have the advantage of being produced inside cells by standard biological means. Designing RNAs is challenging but fun, on a previous blog post a cool RNA structure was shown, but how does the design of such structures work, what are the principles behind it? Keep on reading to get a sneak peek into how we use our laptops and pipettes to design RNA origami nanostructures!
Just like in many other creative initiatives, DNA and RNA origami designs start with a concept, something you wish to build. Now, you have to adapt your idea to the principles that the different architectures have. In the case of DNA origami, which is well illustrated in this post written by Yash a large single-stranded DNA is folded into a specific shape using many small also single-stranded DNAs that bind the large DNA by base complementarity, acting as staples.
The main difference between DNA and RNA origami structures is that the latter are designed and built with only a single-strand, this is a very important feature because allows RNA Origami structures to be generated enzymatically, using the natural transcription process, being able to produce large amounts of them at an affordable price and produce them inside cells to perform intracellular functions.
The other basic principle of this architecture is also shared with DNA Origami, base complementarity. This is the key of nucleic acid nanotechnology, we can design sequences of RNA that have certain parts binding to each other to create 3D structures of many different shapes. Once we have the conceptual 3-dimensional idea, we have to hand-write it in 2 dimensions, we use a standard text-editor software for this task. Then we use another piece of software that is able to convert that text file into a 3D model of the structure, this is an iterative process that is repeated until the 3 D model satisfies our desires. Here, we can get some help and input from computer-based simulations such as oxDNA (or oxRNA in our case), that can provide valuable hints on the structural behavior of our design before we produce it in the lab, for further reading on how these simulations work, check out this post written by Joakim.
The third and last step is to design a sequence that will fit the base-pair constrains specified in our text blueprint, a different piece of software can do that for us.
Now, we are ready to convert that RNA sequence to DNA, basically by substituting the Uracil nucleotide (U) by its DNA counterpart, Thymine (T) and introduce an RNA polymerase promoter sequence, which the RNA polymerase will recognize, bind to and start the synthesis of the RNA from there on. Finally, we will introduce this sequence into a bacterial plasmid in a cloning process, when we have our bacterial DNA plasmid encoding our Origami sequence we will introduce it inside bacteria and use the fast replication abilities of these organisms to produce our RNA Origami inside the bacteria.
Inside the bacteria our Origami can perform multiple tasks, we design them so they can mainly bind to different proteins and arrange them in desired ways to get a higher degree of control of their activities, for what it is called metabolic engineering, we also use them as sensors that are able to detect a range of molecules in vivo.
If you liked this entry, stay tuned to see further cool applications of DNA and RNA nanorobots!
Néstor Sampedro, PhD Student at iNANO, Aarhus University
DNA-Robotics 