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- Creators: School of Life Sciences
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DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.
CRISPR-Cas based DNA precision genome editing tools such as DNA Adenine Base Editors (ABEs) could remedy the majority of human genetic diseases caused by point mutations (aka Single Nucleotide Polymorphisms, SNPs). ABEs were designed by fusing CRISPR-Cas9 and DNA deaminating enzymes. Since there is no natural enzyme able to deaminate adenosine in DNA, the deaminase domain of ABE was evolved from an Escherichia coli tRNA deaminase, EcTadA. Initial rounds of directed evolution resulted in ABE7.10 enzyme (which contains two deaminases EcTadA and TadA7.10 fused to Cas9) which was further evolved to ABE8e containing a single TadA8e and Cas9. The original EcTadA as well as the evolved TadA8e where shown to form homodimers in solution. Although it was shown that tRNA binding pocket in EcTadA is composed by both monomers, the significance of TadA dimerization in either tRNA or DNA deamination has not been demonstrated. Here we explore the role of TadA dimerization on the DNA adenosine deamination activity of ABE8e. We hypothesize that the dimerization of TadA8e is more important for the DNA deamination than for the tRNA deamination. To explore this, I conducted a urea titration on ABE8e to disrupt TadA8e dimerization and performed single turnover kinetics assays to assess DNA deamination rate of ABE8e’s. Results showed that DNA deamination rate and efficiency of ABE8e was already impaired at 4M urea and completely lost at 7M. Unfortunately, CD measurements at the equivalent urea concentrations indicate that the loss of activity is due to the unfolding of ABE8e rather than the disruption of TadA8e’s dimerization.