The initial aim of the APTAFRAME project was to provide a strategy for spatial control over affinity reagent discovery process utilising the power of in vitro evolution in conjunction with the spatial addressability of structural DNA nanotechnology. This was mainly focused to evolve aptamers (nucleic acids that bind specifically to other molecules) against proteins of interest using co-evolution as a driver for cooperative binding. Unfortunately, after the proposal was granted, we learned that the main idea of our project was being exploited in an advanced state by other research group.
We then focused on producing the next generation of evolutionary methods initially envisioned in APTAFRAME, but with the end goal of protein and antibody discovery through ribosome display. This new challenge necessitated the evolution of ribosomes to translate information encoded in mXNAs, as mRNAs displayed on origami frames are highly sensitive to biological and chemical degradation during the in vitro translation step. To reach its main goal there are two objectives to achieve: 1) First develop a new method for ribosome evolution. 2) Implementation of this method to evolve an XNA dependent translation system, 3) use this system to display antibodies on DNA origami frames.
Current methods for ribosome evolution are mainly performed through the pull-down approach. However, this method has several drawbacks associated. Pull-down methods limit the turnover / initiation nature of translation during selection process. Secondly, the recovery of information of the active ribosomes is through reverse transcription, which may lead to mutations or deletions in positions where the 16S rRNA has modifications. To avoid these drawbacks, we implemented a new method that allowed us to split genotype from phenotype, avoiding the need for recovery of information directly from active ribosomes. This method also permits the recording of turnover reaction of ribosomes, since it does not require ribosome stalling for evolution to proceed. Therefore, our new method allows for a true selection of ribosome activity including the initiation and termination steps.
Given the bottleneck encountered during ribosome display of (poly)peptides on DNA origami frames, our initial efforts were focused on developing the new method to evolve an mXNA dependent translation machinery. We have managed to implement most of the elements necessary for our new method to work and the results are promising. However, we have not yet been able to begin selection attempts towards mXNA dependent ribosomes. The impact of the COVID19 pandemic during the granted proposal has slowed down the execution and finalisation of this project.
