Enzyme mediated temporal control of DNA based systems

In the field of DNA Nanotechnology nucleic acids are used as nanoscale materials to rationally engineer nanodevices and nanomachines. When developing such these DNA-based systems a key aspect is to be able to precisely control them. In particular during my PhD, I have focused on the possibility to introduce a temporal control within these artificial systems, mimicking temporal patterns present in biological systems. In Chapter 2, I described in detail the first project that I have carried out during my PhD in which I developed an enzyme-based strategy to temporally control DNA strand displacement reactions (SDR) by introducing a tuneable onset time delay. The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain. Specific enzymatic degradation of the blocker strand subsequently enables SDR. This strategy is extremely versatile and can be extended to multiple class of enzymes thus allowing the temporal control of multiple SDRs in the same solution. Finally, I also demonstrate three possible applications of the delayed SDRs to temporally control: 1) the ligand release from a DNA nanodevice, 2) the inhibition of a target protein by a DNA aptamer, and 3) the output signal generated by a DNA logic circuit. In Chapter 3, I further explore the possibility to temporally control DNAbased reactions, and inspired by naturally occurring temporal regulatory mechanisms, I described a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). I also proved that it is possible to orthogonally delay two different pulse reactions in the same solution. Finally, I reported two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns. In chapter 4 I focused on the development of DNA-based membrane-less synthetic cells able to sustain enzymatic reactions with programmable spatial distribution and temporal evolution. To achieve this, I use nucleases and DNA repair enzymes specifically targeting nucleic-acid substrates, which can be localised within the condensates through base pairing. Finally, I use the reaction-diffusion processes to establish static sub-compartments in the condensates, which can be selectively and individually targeted by enzymes. The results showed in this PhD thesis represent a further step towards a deeper regulation of DNA-based reactions, by introducing a temporal control, that can be used to precisely regulate DNA-based nanomachines and nanostructures.