Bio-inspired strategies to control DNA-based nanodevices and nanostructures

DNA and RNA nanotechnology employ synthetic nucleic acid to engineer and organize structural and functional systems with different levels of complexity (2D DNA nanostructures, DNA origami, tetrahedral DNA, molecular beacons, etc.) at nanoscale. Taking advantage from the high specificity and programmability of the DNA/RNA base-pairings, biocompatibility and the quite easy and inexpensive synthesis, synthetic nucleic acids represent particularly appealing and useful biomaterials with potential sensing and drug delivery applications. Understanding the general design principles and biochemical interactions among proteins, nucleic acids, and all the other molecules that constitute life's building blocks is one of the main aims of synthetic biology. During my PhD I developed several programmable nucleic acid responsive systems and modules able to respond to a wide range of stimuli, including nucleic-acids (i.e., single- or double-stranded DNA or RNA strands), ions and proteins (i.e. antibodies) through innovative natureinspired mechanisms. After an introduction on DNA nanotechnology and cell-free technology, I will focus on the advantages of catalytic nucleic acids (i.e., Ribozymes or DNAzymes) coupled with the possibility to introduce entropically disordered regions to rationally control the folding and thus the activity of a Cu2+-dependent self-cleavable DNAzyme (Chapters 2). I have also exploited one of the intriguing mechanisms with which Nature stores, processes and transfers information in the complex environment of cells by harnessing non-covalent interactions that allow biomolecules to be co-localized in an extremely confined volume. On this regard, in Chapter 3, I will describe a re-created spatial co-localization mechanism of interacting DNA strands to control synthetic reactions for the assembly/disassembly of DNAbased nanostructures by using antibodies. Then, in Chapter 4 I will propose a novel approach to mediate the communication between non otherwise interacting proteins by using a synthetic DNA-based device. Chapter 5 underlines the advantages provided by in vitro transcription to control DNA-based nanoswitches and nanostructures.