Digital control of protein-nucleic acid interactions with self-assembled DNA nanosystems

Understanding how enzymes access, transform or degrade a nucleic acid nanostructure is an essential step for the progress of functional DNA nanotechnology. Most approaches for the analysis of nucleic acids require enzymatic reactions. For instance sequencing-by-synthesis of confined DNA molecules is a most established approach for the analysis of the entire genomic information in living organisms. Little is known however about the physical factors that regulate enzymes functions in highly confined systems. In this doctoral thesis, I have investigated the activity of type II restriction enzymes (REs) on basic, two-dimensional DNA nanostructures (DNA origami, i.e. triangles and rectangles) that are formed upon the spontaneous hybridization of many single stranded (ss)DNA molecules over the 7,249-nt-long M13mp18 ssDNA phage genome that serves as scaffold for the inherent DNA self-assembly process. The primary action of REs is DNA fragmentation, and they are a primary defensive mechanism to viral infection in bacteria. Type II REs have an exquisite ability to specifically recognize dsDNA binding sites (termed restriction sites) and irreversibly cleave the inherent DNA molecules within the sites. They are in addition essential tools for DNA cloning in current bioengineering and biotechnology applications. On the other hand, the M13mp18 scaffold naturally possesses a number of restriction sites for each of several, known restriction enzymes. In addition, DNA origami are comprised of different structural motifs that are responsible for molecular confinement of the involved DNA molecules and the peculiar mechanical properties of the shape. My work primarily consists of an unprecedented investigation of the action of >10 type II restriction enzymes on two-dimensional (2D) DNA origami. For two enzymes in particular (HhaI and Hin1II) we fully mapped the site-specific action by activating one site at a time (by generating DNA origami mutants), and measuring the fragmentation pattern of the DNA scaffold by gel electrophoresis, after melting the DNA nanostructure. With such mutational analysis of 2D DNA origami we found that (a) restriction reactions can be efficiently inhibited in 2D DNA origami, while similar inhibition cannot be achieved with the corresponding unfolded dsDNA scaffold (as expected). We argue that the observed behaviour of dense nucleic acid architectures naturally emerges as a result of reduction in spatial degrees of freedom near restriction sites, which can be controlled through small changes to the degree of mechanical stress (e.g. torsion) near the sites. (b) In 2D DNA origami, the action of REs on a site can be predicted from the structure of the three 16 bp-long adjacent dsDNA segments, with the site located in the central one (16 bp is the distance between two consecutive crossovers that join the dsDNA segments). Specifically, a site can be cleaved only if (c) it is located ≥4 bp from a crossover junction, and (d) near the site, each of the adjacent dsDNA segments presents a nick in one of the two strands that form the duplex. This quantitative study reveals therefore that restriction enzyme action can be digitally controlled with the closest neighbouring 2D DNA structural pattern surrounding a restriction site. These unprecedented results also suggest how to design functional nucleic acid nanostructures, with important implications for the implementation of innovative nano-biosensor, that is briefly anticipated in the concluding section of this thesis.