High-Throughput Screening for Oligonucleotide Detection by ADE-OPI-MS

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Biotechnology

Background:

• Oligonucleotides are crucial in biomedicine, but high-throughput analysis remains a challenge.
• Existing methods for oligonucleotide detection and analysis lack efficiency for large-scale studies.
• Terminal deoxynucleotide transferase (TdT) plays a key role in DNA synthesis.

Purpose of the Study:

• To develop a high-throughput analysis method for oligonucleotides.
• To apply this method for engineering terminal deoxynucleotide transferase (TdT) with enhanced catalytic activity.
• To improve the efficiency of DNA synthesis through enzyme engineering.

Main Methods:

• Development of a high-throughput oligonucleotide analysis method using acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS).
• Application of the ADE-OPI-MS method to screen approximately 10,000 TdT mutants for enzymatic activity.
• Integration of identified high-activity TdT variants to enhance catalytic function.

Main Results:

• The developed ADE-OPI-MS method achieved an analysis rate of 3 seconds per sample, a 60-fold increase in efficiency over gel analysis.
• Three novel TdT variants with significantly higher catalytic activities were identified.
• The catalytic activity of TdT was improved approximately 4-fold by integrating the engineered mutants.

Conclusions:

• A robust high-throughput screening method for oligonucleotide analysis was successfully established.
• The engineered TdT variants provide a foundation for *ab initio* DNA synthesis.
• This work opens avenues for broader applications of oligonucleotide analysis in biomedical research.