Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers

Area of Science:

• Synthetic Biology
• Biochemistry
• Molecular Biology

Background:

• Xenobiotic nucleic acids (XNAs) show potential as genetic materials but face challenges in in vivo applications, primarily due to lack of orthogonality.
• Orthogonality requires XNAs that do not interact with natural nucleic acids or enzymes, and vice versa, alongside specialized XNA-processing enzymes.

Purpose of the Study:

• To report a novel XNA, 3 -2 phosphonomethyl-threosyl nucleic acid (tPhoNA), as a candidate for in vivo genetic material applications.
• To demonstrate the synthesis and viability of tPhoNA as a genetic material, assessing its orthogonality and compatibility with enzymatic processes.

Main Methods:

• Chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks.
• Demonstration of DNA duplex destabilization upon tPhoNA incorporation.
• Engineering of a novel tPhoNA synthetase and utilization of an XNA reverse transcriptase.
• In vivo orthogonality testing in E. coli.

Main Results:

• Established chemical synthesis routes for tPhoNA and its building blocks.
• Showed that tPhoNA destabilizes DNA duplexes.
• Demonstrated tPhoNA as a viable genetic material with an aggregate error rate of ~17 × 10^-3 per base, compatible with functional XNA isolation.
• Confirmed limited interaction of E. coli cellular machinery with tPhoNA genetic information.

Conclusions:

• 3 -2 phosphonomethyl-threosyl nucleic acid (tPhoNA) is a novel XNA with modified sugar and phosphate backbone, representing a significant advance in biorthogonality.
• tPhoNA shows promise for in vivo applications, demonstrating essential characteristics for synthetic genetic systems.
• This work paves the way for introducing XNA systems in vivo, expanding the possibilities of genetic material beyond natural DNA and RNA.