Periodic Reporting for period 1 - APTAFRAME (DNA-origami frame platform for co-evolution ligand selection)
Okres sprawozdawczy: 2019-04-01 do 2021-03-31
The humoral immune system is a key aspect of human biology in fighting off diseases and eliminating pathogens. Furthermore, engineered antibodies can fight an increasing number of diseases including cancer and autoimmune conditions. Therefore, strategies to systematically and efficiently generate high-affinity antibodies are important parts of modern biotechnology. However, methods of rapidly creating antibodies binding to a very specific region of a target remains challenging and a major unmet goal.
New methods of selection, based on spatial control over affinity reagent discovery process should therefore accelerate and increase efficiency of antibody discovery. The overall aim of APTAFRAME was to build a novel technology platform for the systematic discovery of ligands leveraging cooperative binding effects in the context of spatially configurable DNA origami frameworks and to initially demonstrate this in the context of nucleic acid aptamer libraries. As a next step, APTAFRAME envisaged to evolve aptamers composed of macrocyclic peptides (MCP) and antibodies against proteins of interest using co-evolution as a driver for cooperative binding. The best method to display peptides on the DNA origami frames is through ribosome display. Protein discovery through ribosome display relies on the reading of a messenger RNA (mRNA) that also serves as sequence-to-protein connector during high-throughput readout. Unfortunately, the chemical (and biological) degradability of RNA poses a challenge for the development of this, and similar methodologies. Xenonucleic acids (XNAs) are alternative informational polymers largely resistant to chemical and biological degradation. Unfortunately, current ribosomes cannot efficiently translate peptides and proteins from mXNAs. We have therefore focused part of our efforts trying to circumvent this problem by creating ribosomes that can read genetic information encoded in mXNAs. Our hope is that by producing mXNA dependent ribosomes we will create a robust system for the decoration of DNA nano-objects with translated peptides and proteins utilised as a framework for cooperative evolution.
New methods of selection, based on spatial control over affinity reagent discovery process should therefore accelerate and increase efficiency of antibody discovery. The overall aim of APTAFRAME was to build a novel technology platform for the systematic discovery of ligands leveraging cooperative binding effects in the context of spatially configurable DNA origami frameworks and to initially demonstrate this in the context of nucleic acid aptamer libraries. As a next step, APTAFRAME envisaged to evolve aptamers composed of macrocyclic peptides (MCP) and antibodies against proteins of interest using co-evolution as a driver for cooperative binding. The best method to display peptides on the DNA origami frames is through ribosome display. Protein discovery through ribosome display relies on the reading of a messenger RNA (mRNA) that also serves as sequence-to-protein connector during high-throughput readout. Unfortunately, the chemical (and biological) degradability of RNA poses a challenge for the development of this, and similar methodologies. Xenonucleic acids (XNAs) are alternative informational polymers largely resistant to chemical and biological degradation. Unfortunately, current ribosomes cannot efficiently translate peptides and proteins from mXNAs. We have therefore focused part of our efforts trying to circumvent this problem by creating ribosomes that can read genetic information encoded in mXNAs. Our hope is that by producing mXNA dependent ribosomes we will create a robust system for the decoration of DNA nano-objects with translated peptides and proteins utilised as a framework for cooperative evolution.
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.
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.
We have worked towards the creation of a technology platform for protein and antibody discovery through ribosome display on DNA origami frames. Due to mRNA degradation during in vitro translation, we have focused our efforts to implement mXNA dependent ribosomes. We have worked towards a method for ribosome evolution which circumvents major bottlenecks of current pull-down approaches, which will be used to evolve mXNA dependent ribosomes. Our new method allows turnover and translation initiation to be part of the ribosome evolution process and bypasses potential artefacts occurring during genetic information recovery from the selected ribosomes. Moreover, our new method is more suited for function enhancement of ribosomes that contain certain initial activity, since we can precisely fine-tune the stringency of the evolution process. Once completed, the new ribosomes will be used to display peptides, and select for protein based cooptamers in the context of the spatially addressable DNA origami platform technology.
Selection of ribosomes that read out genetic information from mXNA is currently under way. When we succeed, these ribosomes will be implemented within the context of the spatially addressable DNA origami platform technology. This new ribosome should help to overcome mRNA degradation— one of the main bottlenecks associated with these technologies; the new ribosomes should allow stable display and decoration of DNA origami frames with antibody and peptide ligands, enabling a wide range of applications in both biotechnology and medicine.
Selection of ribosomes that read out genetic information from mXNA is currently under way. When we succeed, these ribosomes will be implemented within the context of the spatially addressable DNA origami platform technology. This new ribosome should help to overcome mRNA degradation— one of the main bottlenecks associated with these technologies; the new ribosomes should allow stable display and decoration of DNA origami frames with antibody and peptide ligands, enabling a wide range of applications in both biotechnology and medicine.