Artificial nucleotide codons for enzymatic DNA synthesis.

Area of Science:

• Synthetic biology
• Nucleic acid chemistry
• Biotechnology

Background:

• Enzymatic DNA synthesis traditionally uses single nucleotides.
• Producing complex nucleic acid structures like xenonucleic acids (XNAs) is challenging.
• Developing novel nucleotide building blocks is crucial for expanding synthetic biology.

Purpose of the Study:

• To report a high-yielding solid-phase synthesis method for trinucleotide triphosphates (dN3TPs).
• To demonstrate the utility of these dN3TPs in enzymatic DNA synthesis.
• To enable the production of XNAs and facilitate in vitro selection of modified nucleic acids.

Main Methods:

• Solid-phase synthesis of unmodified and modified dN3TPs.
• Stabilization of dN3TP scaffolds using phosphorothioate units.
• Enzymatic DNA synthesis utilizing the synthesized dN3TPs.

Main Results:

• Successful high-yielding synthesis of dN3TPs.
• Demonstrated enzymatic incorporation of dN3TPs into DNA strands when scaffolds are phosphorothioate-stabilized.
• Established a method for using three-letter nucleotide systems in enzymatic synthesis.

Conclusions:

• Solid-phase synthesis provides an efficient route to dN3TPs.
• Phosphorothioate stabilization is key for enzymatic use of dN3TPs.
• This approach advances the synthesis of XNAs and the selection of functional nucleic acids.