De novo DNA synthesis using polymerase-nucleotide conjugates

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Chemistry

Background:

• Traditional oligonucleotide synthesis relies on the nucleoside phosphoramidite method.
• This method has limitations, including a synthesis length of approximately 200 bases and the generation of hazardous waste.
• There is a need for alternative, more efficient, and environmentally friendly oligonucleotide synthesis strategies.

Purpose of the Study:

• To develop a novel enzymatic strategy for oligonucleotide synthesis.
• To utilize terminal deoxynucleotidyl transferase (TdT) for template-independent nucleotide incorporation.
• To establish a method for controlled, sequential addition of nucleotides to create defined oligonucleotide sequences.

Main Methods:

• Conjugating individual deoxyribonucleoside triphosphate (dNTP) molecules to terminal deoxynucleotidyl transferase (TdT) enzymes.
• Utilizing the TdT-dNTP conjugate to sequentially incorporate a single nucleotide into a primer.
• Cleaving the covalent linkage between the TdT and the incorporated nucleotide to allow for subsequent extension steps.
• Iterating the process to build defined oligonucleotide sequences.

Main Results:

• Demonstrated quantitative single-nucleotide extension of a primer by TdT-dNTP conjugates within 10-20 seconds.
• Showcased the iterative capability of the method to synthesize defined oligonucleotide sequences.
• Established a proof-of-concept for an enzymatic oligonucleotide synthesizer.

Conclusions:

• The developed TdT-dNTP conjugate strategy offers a promising new method for oligonucleotide synthesis.
• This enzymatic approach overcomes limitations of traditional phosphoramidite synthesis, potentially enabling longer and greener synthesis.
• The findings lay the groundwork for the development of a novel enzymatic oligonucleotide synthesizer.