Template-Independent Enzymatic RNA Synthesis

Area of Science:

• Biochemistry
• Synthetic Biology
• Molecular Biology

Background:

• Traditional RNA synthesis methods, such as phosphoramidite synthesis, often involve hazardous reagents and sensitive conditions.
• Developing efficient and controlled methods for de novo RNA synthesis is crucial for various biotechnological applications.

Purpose of the Study:

• To report a novel route for preparing ribonucleoside triphosphates with a removable 3 ahydroxy (3 ahydroxy) blocking group.
• To demonstrate template-independent enzymatic RNA synthesis (TIERS) using engineered DNA polymerases and these modified triphosphates.
• To establish a cyclic reversible termination framework for controlled, stepwise RNA elongation.

Main Methods:

• Synthesis of ribonucleoside triphosphates featuring a 3 ahydroxy (3 ahydroxy) group.
• Enzymatic assays using human DNA polymerase theta (Polθ) and mimiviral PrimPol with the modified triphosphates.
• Demonstration of cyclic RNA synthesis through repeated nucleotide addition and 3 ahydroxy group cleavage.

Main Results:

• Two DNA polymerases, Polθ and PrimPol, were shown to accept the 3 ahydroxy-modified triphosphates as substrates.
• Cleavage of the O-N bond in the 3 ahydroxy group successfully regenerated the 3 ahydroxy hydroxyl group, enabling subsequent nucleotide addition.
• Successful stepwise addition of three ribonucleotides in three cycles using engineered Polθ demonstrated the feasibility of the TIERS process.
• The method enables de novo RNA synthesis without hazardous solvents or sensitive reagents.

Conclusions:

• A novel and efficient method for template-independent enzymatic RNA synthesis (TIERS) has been established.
• The cyclic reversible termination framework using 3 ahydroxy-modified triphosphates offers a simplified and safer alternative to phosphoramidite RNA synthesis.
• This approach is amenable to instrument adaptation and holds promise for the de novo synthesis of RNA with defined sequences.