Single-stranded DNA : methods and application in nanotechnology

Basic molecular and cell research, production of recombinant proteins, diagnostic detection of genetic mutations, construction of nanostructures and high-throughput DNA sequencing are only a few examples of the diverse set of applications that are amenable thanks to the availability of synthetic DNA polymers in biomedicine. Strategic investments and technical progress together with the introduction of automation in the synthesis of DNA oligomers enabled to transform, in just a few decades, a process mastered only by a niche of biochemists into an affordable and available custom-made product to every scientific field. Despite such progress, innovation soon reached a plateau due to intrinsic limitations of the synthesis process, putting a barrier at two hundred nucleotides as the maximum length of synthetic DNA molecules. In the meanwhile, molecular biologists closed the gap thanks to a better understanding of polymerases and the mastering of directed evolution protocols making it possible to redesign processes that are more similar to what happens in nature, taking advantage of existing and improved enzymes for the generation of long and high-quality DNA molecules. This enabled to find novel applications for DNA such as gene editing or information storage.

In this thesis I focused on the enzymatic production and functionalization of single stranded DNA. More specifically, in paper I we directed our attention to optimize the protocol for the templated enzymatic synthesis of oligonucleotides. We highlighted possible limitations of the technique and proposed a solution in employing a single stranded binding protein greatly decreasing double stranded DNA contaminants. In paper II we further extended the workflow. In here, we focused on continuing the previous protocol to accommodate the production of chimeric DNA-protein molecular tools needed in nanotechnology where DNA is considered more a building material rather than an information rich polymer while the actuation of a particular function is operated by proteins. We worked on a minimal bacteria-derived self-tagging domain that has the capacity to establish a covalent bond with a specific DNA sequence and some applications are suggested.

Paper III represents the natural extension of this work even if, in this specific case, the earlier presented rational is reverted. More specifically, a biosensor for the detection of aquatic microorganisms was produced with the characterized bioconjugation technique where the chimeric protein was used as recognition moiety and the oligonucleotide as signal amplification device through its intrinsic DNAzyme activity. Finally, in Paper IV, we decided to use all the previously gathered knowledge – enzymatic DNA production and bioconjugation techniques – to conceive a novel basic biology investigation tool for the study of spatial organization of proteins. Here we took advantage of the possibility to grow a localized and unique DNA polymer with the ability to target proteins with DNA-protein chimeras. The resulting product is then recovered and decoded by next generation sequencing.

List of scientific papers

I. Ducani C., Bernardinelli G., & Högberg B. (2014). Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA. Nucleic Acids Research. 42(16), 10596–10604.
https://doi.org/10.1093/nar/gku737

II. Bernardinelli G., & Högberg B. (2017). Entirely enzymatic nanofabrication of DNA-protein conjugates. Nucleic Acids Research. 45(18), e160.
https://doi.org/10.1093/nar/gkx707

III. Bernardinelli G., Oloketuyi S., Werner S. W., Mazzega E., Högberg B., de Marco A. (2019) A compact nanobody-DNAzyme conjugate enables antigen detection and signal amplification. [Manuscript]

IV. Bernardinelli G., Werner S. W., Rocamonde-Lago I., Hoffecker I. T., Högberg B. (2019). RollingTag-seq: a microscopy-free DNA sequencing based approach for protein spatial organization probing. [Manuscript]