Final Report Summary - XNA (Development of an artificial information system)
Nucleic acids that are produced in vivo for therapeutic, diagnostic and other health and environment-related purposes, are restricted to DNA and RNA. Therapeutic molecules made of DNA and RNA are endowed with low biostability and poor pharmacodynamics, while exposing human cells to the risk of genetic transformation. Therefore, we have propagated additional type of nucleic acids (XNA), which enlarges the scope of drug discovery through conventional biotechnology and to prevent dissemination of genetic information in the human body and natural habitats.
For this purpose, several artificial nucleic acids have been synthesized, and biophysical and biochemical analyzed for their potential to exchange information with DNA and RNA. In bacteria we have replaced DNA at the level of several codons with XNA and discovered that this XNA can still be transliterated by the bacterial replication system. We have also demonstrated that, in bacteria, the DNA of a whole gene can be replaced by XNA, which can still code for a functional protein. This means that the four canonical DNA letters (A, G, C, T) can be replaced in vivo by other letters, without disturbing the genetic information system. It also demonstrates that full orthogonal information systems (an information system that does not communicate with DNA and RNA) can be obtained by changing the backbone and base moiety of the natural nucleic acids. In order to endow such information system with a metabolic safety level, the metabolic precursors for XNA have been chemically changed and an uptake system for metabolic precursors in bacteria has been elaborated.
The chemical selection approach has led to the identification of the most ideal XNA for in vitro and in vivo application. This XNA is chemically and enzymatically stable, does recognize itself but recognize poorly DNA, is non-toxic, can be synthesized by evolved polymerases, is metabolically easily accessible because of the presence of a phosphonate function and represents the first acyclic XNA that is recognized in vivo. It has been evaluated using a binary coding system (based on 2 nucleobases instead of 4 nucleobases).
We have realized the conversion of DNA information to XNA and back to DNA using evolved polymerases. This exchange of information has been used to develop therapeutic aptamers consisting of XNA and targeting BACE-1 and VEGF. It demonstrates the power of this selection technology to discover new and enzymatically stable aptamer drugs.
In conclusion, we have elaborated several technological gaps between the chemical capability of synthesizing variant XNA and the biosynthetic availability of replicating enzymes able to replicate such chemical modified nucleic acids, which leads to potential new application in medicine and biotechnology
For this purpose, several artificial nucleic acids have been synthesized, and biophysical and biochemical analyzed for their potential to exchange information with DNA and RNA. In bacteria we have replaced DNA at the level of several codons with XNA and discovered that this XNA can still be transliterated by the bacterial replication system. We have also demonstrated that, in bacteria, the DNA of a whole gene can be replaced by XNA, which can still code for a functional protein. This means that the four canonical DNA letters (A, G, C, T) can be replaced in vivo by other letters, without disturbing the genetic information system. It also demonstrates that full orthogonal information systems (an information system that does not communicate with DNA and RNA) can be obtained by changing the backbone and base moiety of the natural nucleic acids. In order to endow such information system with a metabolic safety level, the metabolic precursors for XNA have been chemically changed and an uptake system for metabolic precursors in bacteria has been elaborated.
The chemical selection approach has led to the identification of the most ideal XNA for in vitro and in vivo application. This XNA is chemically and enzymatically stable, does recognize itself but recognize poorly DNA, is non-toxic, can be synthesized by evolved polymerases, is metabolically easily accessible because of the presence of a phosphonate function and represents the first acyclic XNA that is recognized in vivo. It has been evaluated using a binary coding system (based on 2 nucleobases instead of 4 nucleobases).
We have realized the conversion of DNA information to XNA and back to DNA using evolved polymerases. This exchange of information has been used to develop therapeutic aptamers consisting of XNA and targeting BACE-1 and VEGF. It demonstrates the power of this selection technology to discover new and enzymatically stable aptamer drugs.
In conclusion, we have elaborated several technological gaps between the chemical capability of synthesizing variant XNA and the biosynthetic availability of replicating enzymes able to replicate such chemical modified nucleic acids, which leads to potential new application in medicine and biotechnology