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Rotaxane-Oligonucleotides: Stimuli-Responsive Mechanically Interlocked Architectures to Control Oligonucleotide Activity and Gene Transcription

Periodic Reporting for period 1 - Rotaxane-DNA (Rotaxane-Oligonucleotides: Stimuli-Responsive Mechanically Interlocked Architectures to Control Oligonucleotide Activity and Gene Transcription)

Reporting period: 2018-06-07 to 2020-10-06

During this MSCA, I helped develop a new type of synthetic but biocompatible DNA. The key innovative feature of my work was that the DNA molecule was wrapped in a circular molecule to create a structure called a rotaxanes. The key challenge I addressed in my project was how to make this rotaxane-DNA and a preliminary investigation of its properties. I demonstrated that the ring around the DNA changes its biological properties and developed new rings that can be used to change the properties of rotaxane-DNA further. This rotaxane-DNA is an exciting platform for development of tools for studying the behaviour of cells and new therapeutic molecules based on DNA and RNA. The overall objectives of the project, which will continue beyond my project, are to deliver rotaxane-DNA and RNA that can be used to control protein expression in cells and whole organisms.
I prepared modified single DNA bases for their incorporation into solid phase oligonucleotide synthesis, and then used these short alkyne and azide terminated oligonucleotide fragments to prepare either the free axle (via traditional 'click' chemistry) or the interlocked rotaxane-DNA (via AT-CuAAC). Through systematic optimization, I was able to achieve these new materials in good yield with high selectivity (100% conversion to the interlocked structure). These materials were tested as primers for PCR, evaluated for their duplex formation by CD spectroscopy, and initial degradation experiments were performed with exonucleases to further probe the stabilizing effect of the mechanical bond. A significant amount of work went into optimizing the purification, isolation and analysis of these materials, as well as finding mild methods to remove Cu (necessary for the formation of the interlocked structure, though known to be highly cytotoxic). Cleavable linkers were tested for their overall compatibility with macrocycle formation (Ni mediated macrocylization), and there is on-going work in the Goldup lab to generate novel cleavable macrocycles, responsive to selected stimuli (e.g. photolabile), which will help further work begun during this MSCA.

The results of this study formed the basis of a successful application for further funding which will be used to take these new molecules towards applications in chemical biology. The work also formed the basis of a publication in J. Am. Chem. Soc. and several presentations at national meetings in the UK.
When I began the project my hosts had preliminary data showing that the synthesis of rotaxane-DNA was possible but the methods needed further development. During the MCSA I developed a reliable procedure for the synthesis of rotaxane-DNA and analytical techniques to determine the outcome of the reaction. I also investigated new types of rings for inclusion in the rotaxane-DNA structure.

In future, my hosts will develop these methods further to incorporate RNA in place of DNA and systems in which the effect of the ring can be controlled. The work during this MCSA provides a foundation for these future developments as I have demonstrated the long term potential of rotaxane-oligonucleotides for a cross section of current synthetic oligonucleotide applications and drive future applications of these materials towards a versatile platform for chemical biology.
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