Toward in vivo nanostructures: hybrid origami folded on De Bruijn sequence scaffolds

by Alessandro Ceccarelli

16:00 (40 min) in Daysh G.07

The last 20 years witnessed the rise of nucleic acid engineering, which became one of the most promising technologies for the immediate future. Started by Rothemund's first origami, nanobioengineering now shows a series of interesting features highly relevant in many fields: bio-compatibility, ease of replication and ease of further functionalizing.

Today the horizon of applications is still widening, especially concerning the complexity of the structures designed, and many projects are aiming at the development of diagnostic or therapeutic nanodevices. The most relevant steps forward are well represented by drug delivery devices, synthetic nanopores and origami arrays.

It is natural to think that soon new nanodevices will be designed to be assembled, to interact and to execute their functions within the living cell. Preliminary studies already demonstrated that DNA origami and RNA assemblies are compatible with the cellular environment, but it remains unknown if the introduction of exogenous nucleic acids in a cell will have unpredictable consequences.

For DNA origami in particular, the main sources of nucleic acids are bacteriophages and bacteria for the easiness of replication and extraction of single stranded DNA. This obviously raises many doubts over the suitability of such materials for clinical applications, especially in human medicine.

This background shows clearly how a bio-orthogonal sequence is needed to accompany the transition of bioengineering from "in vitro" to "in vivo", to overcome the doubts mentioned before and to provide a reliable tool for every field of synthetic biology. A fully synthetic sequence can cover multiple roles, in addition to the origami scaffold it can be used as spacer in synthetic circuits, as structural part in DNA probes and linkers and in all roles that require a complete bio-inert component.

In the lab, in vitro, we have successfully assembled a square DNA origami from a 2.4 kilobase DBS, and a triangle RNA-DNA hybrid origami from a 1 kilobase RNA DBS. These structures are approx. 50nm and 30nm under AFM, respectively. To verify bio-orthogonality we have started preliminary investigations, so far demonstrating that our RNA DBS scaffold is not degraded inside E.coli cells over an incubation period of 40 minutes. With our work we also demonstrate how a fully synthetic scaffold can fold an origami in a life-compatible temperature range.