A Universal Nucleoside-to-Nucleoside-5'-Triphosphate Enzyme Cascade Driven by Polyphosphate

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzymology

Background:

• Nucleoside-5'-triphosphates (NTPs) are essential for nucleic acid synthesis.
• Chemical synthesis of NTPs faces challenges, particularly low yields in the final 5'-triphosphorylation step.
• Efficient and scalable methods for NTP production are crucial for research and therapeutic applications.

Purpose of the Study:

• To develop a novel enzymatic cascade for efficient conversion of nucleosides to NTPs.
• To enable the synthesis of both canonical and modified NTPs using a cost-effective and scalable approach.
• To demonstrate the utility of the cascade for producing key molecules for mRNA therapeutics.

Main Methods:

• An enzymatic cascade reaction was designed using two enzymes: deoxynucleoside kinase (Dm-dNK) and polyphosphate kinase (EbPPK).
• The cascade operates in a single one-pot reaction driven by sodium polyphosphate (polyP) and requires only catalytic amounts of ATP.
• The system was tested for the synthesis of canonical rNTPs and dNTPs, as well as eleven modified nucleotide analogues.

Main Results:

• The two-enzyme cascade successfully converted nucleosides to all canonical rNTPs and dNTPs.
• The reaction demonstrated high efficiency with minimal ATP requirement (≤0.0001 equivalents).
• The cascade produced various modified NTP analogues, including 2'-fluoro-2'-dNTPs, base-modified nucleotides, and N1-methyl-ΨTP (m¹ΨTP) on a milligram scale.

Conclusions:

• A novel, efficient, and cost-effective enzymatic cascade for NTP synthesis from nucleosides has been established.
• This method overcomes limitations of traditional chemical synthesis, offering a scalable route to canonical and modified NTPs.
• The demonstrated production of m¹ΨTP highlights the potential of this cascade for manufacturing essential components for mRNA therapeutics.