Periodic Reporting for period 1 - OLIGOBINPRO (Non-canonical nucleoside incorporation into synthetic RNA-oligonucleotides: investigations towards the discovery of selective RNA-binding proteins)
Berichtszeitraum: 2015-06-01 bis 2017-05-31
The increase of life expectancy around the world comes at the unavoidable cost of a rise in the occurrence of neurological diseases such as Parkinson’s as well as debilitating ones like cancer. These conditions prevent the possibility of a sustained quality of life and cost society vast amounts of money for the treatment and care of patients. It is the responsibility of scientists to find innovative methods for treating and eliminating these diseases, allowing for a healthier quality of life. Recently, RNA-binding proteins (RBPs) have been linked to biological processes leading to these diseases as well as the expression of genes relating to obesity. A plethora of natural chemical modifications in RNA fine-tune its structure, allowing for specific interactions which in turn regulate processes such as gene expression and the tuning of translation. Although these have been known for years, their binding proteins have not been systematically studied. The difficulty of the chemical synthesis of modified oligoribonucleotides (ORNs) represents the major bottleneck in this field. The overall aim of this project is to discover RNA-binding proteins which bind to natural, non-canonical modifications which were identified in the messenger RNA (mRNA) of human cancer cell lines.
In order to deepen our understanding of these modifications, these need to be synthesised and then incorporated into oligoribonucleotide strands mimicking natural mRNA. By incubating with cellular extracts, we aim to identify their binding proteins by analysing them using mass spectrometric techniques. This project aims to set the foundation for the better understanding of the mechanisms responsible for the aforementioned conditions, which will in the long term lead to the design of new therapies for their prevention.
During the course of this fellowship, advancements have been made in the design and synthesis of non-canonical RNA modifications. These have been incorporated into nature mimicking RNA strands which have allowed us to identify RBPs directly associated with them. Further investigation into the identity and functionality of these RBPs, in relation to cancer and epigenetic gene expression is necessary to begin unravelling their specific role in nature. Although only preliminary proteomic results have been obtained at the current stage, the tools developed during this fellowship will enable future scientific investigations and hopefully open new directions with respect to therapy development of relevant diseases.
In order to deepen our understanding of these modifications, these need to be synthesised and then incorporated into oligoribonucleotide strands mimicking natural mRNA. By incubating with cellular extracts, we aim to identify their binding proteins by analysing them using mass spectrometric techniques. This project aims to set the foundation for the better understanding of the mechanisms responsible for the aforementioned conditions, which will in the long term lead to the design of new therapies for their prevention.
During the course of this fellowship, advancements have been made in the design and synthesis of non-canonical RNA modifications. These have been incorporated into nature mimicking RNA strands which have allowed us to identify RBPs directly associated with them. Further investigation into the identity and functionality of these RBPs, in relation to cancer and epigenetic gene expression is necessary to begin unravelling their specific role in nature. Although only preliminary proteomic results have been obtained at the current stage, the tools developed during this fellowship will enable future scientific investigations and hopefully open new directions with respect to therapy development of relevant diseases.
From the beginning of the project, we needed to ensure an up-to-date investigation of RNA binding proteins associated with specific non-canonical modifications. For the purposes of this project, a group of non-canonical RNA modifications was selected to be investigated based on recent literature as well as in-house knowledge of their abundance or rarity in specific human cell lines.
The in-depth investigation of our selected modifications begun with the synthesis of the natural nucleosides as well as their isotopologues. By having both these sets of standards we are now able to carry out in-house stable-isotope dilution methodologies which allow us to quantify these modifications in various human and murine cells as well as in different animal tissues with the help of our collaborators.
Following the synthesis of standards for the purposes of quantification, we continued by pursuing to incorporate our selected modifications at specific loci within RNA strands using solid-phase synthesis. In order to be able to achieve this, appropriately protected phosphoramidites (PA) were designed and synthesised for each of our modifications.
Subsequently, we validated their applicability for the purposes of RNA synthesis and successfully achieved the solid-phase synthesis, deprotection and purification of RNA strands containing all the relevant non-canonical modifications.
In an attempt to probe and begin understanding their role in biological systems, we set out to design and synthesise RNA strands with a specific sequence context which were inspired from published next generation sequencing data. Basing our investigations on this data, we synthesised pairs of "nature mimicking oligoribonucleotides" (modified and unmodified/control strands) which were used as baits for proteomics experiments. By incubating our synthesised RNA strands in cell lysate of cancer cell lines, we are able to "fish-out" proteins which associate/bind to these modifications; or proteins which are repelled by them and instead bind to the canonical/control strands.
The results stemming from this project have been presented in a poster and oral presentation format and will be published in peer reviewed journals in due course.
The in-depth investigation of our selected modifications begun with the synthesis of the natural nucleosides as well as their isotopologues. By having both these sets of standards we are now able to carry out in-house stable-isotope dilution methodologies which allow us to quantify these modifications in various human and murine cells as well as in different animal tissues with the help of our collaborators.
Following the synthesis of standards for the purposes of quantification, we continued by pursuing to incorporate our selected modifications at specific loci within RNA strands using solid-phase synthesis. In order to be able to achieve this, appropriately protected phosphoramidites (PA) were designed and synthesised for each of our modifications.
Subsequently, we validated their applicability for the purposes of RNA synthesis and successfully achieved the solid-phase synthesis, deprotection and purification of RNA strands containing all the relevant non-canonical modifications.
In an attempt to probe and begin understanding their role in biological systems, we set out to design and synthesise RNA strands with a specific sequence context which were inspired from published next generation sequencing data. Basing our investigations on this data, we synthesised pairs of "nature mimicking oligoribonucleotides" (modified and unmodified/control strands) which were used as baits for proteomics experiments. By incubating our synthesised RNA strands in cell lysate of cancer cell lines, we are able to "fish-out" proteins which associate/bind to these modifications; or proteins which are repelled by them and instead bind to the canonical/control strands.
The results stemming from this project have been presented in a poster and oral presentation format and will be published in peer reviewed journals in due course.
The work carried out during this project includes the synthesis of monomers of the non-canonical modifications which allow for:
a) The quantification of these modifications in biological systems (nucleosides and their isotopologues)
b) The incorporation of these modifications into synthesised RNA strands (phosphoramidite building blocks)
These molecules are currently being used for the purposes of the completion of the OLIGOBINPRO project. However, they will also be used for related projects in the future, especially with regards to quantifying RNA modifications in different cell types and tissues. Furthermore, the synthetic route to the PA building blocks has provided indispensable training to the fellow with regards to their synthesis and design.
The most valuable aspect of this project involves the identification of the "writer, reader and eraser" enzymes of these modifications which perhaps govern detrimental biological processes in cancer cells. By identifying them, we hope to inspire further work which may involve the pursuit of small molecule inhibitors to these proteins for the development of new drugs for cancer therapeutics.
Aside from the scientific impact this project will have to the understanding and treatment of cancer, it has also inspired and assisted the fellow to pursue a career in the treatment of cancer. The fellow has recently begun working in a leading pharmaceutical company based in the European Union.
a) The quantification of these modifications in biological systems (nucleosides and their isotopologues)
b) The incorporation of these modifications into synthesised RNA strands (phosphoramidite building blocks)
These molecules are currently being used for the purposes of the completion of the OLIGOBINPRO project. However, they will also be used for related projects in the future, especially with regards to quantifying RNA modifications in different cell types and tissues. Furthermore, the synthetic route to the PA building blocks has provided indispensable training to the fellow with regards to their synthesis and design.
The most valuable aspect of this project involves the identification of the "writer, reader and eraser" enzymes of these modifications which perhaps govern detrimental biological processes in cancer cells. By identifying them, we hope to inspire further work which may involve the pursuit of small molecule inhibitors to these proteins for the development of new drugs for cancer therapeutics.
Aside from the scientific impact this project will have to the understanding and treatment of cancer, it has also inspired and assisted the fellow to pursue a career in the treatment of cancer. The fellow has recently begun working in a leading pharmaceutical company based in the European Union.