Electronically controlled deprotection chemistry for multiplex enzymatic DNA synthesis on a chip with single-base...

Area of Science:

• Biotechnology
• Synthetic Biology
• Materials Science

Background:

• Enzymatic DNA synthesis (EDS) offers an eco-friendly alternative to chemical DNA synthesis for producing long and complex DNA sequences.
• Current EDS methods face challenges in achieving precise sequence control at high throughput.
• Developing automated and scalable DNA manufacturing technologies is crucial for various biological applications.

Purpose of the Study:

• To develop a novel platform for high-throughput, multiplexed enzymatic DNA synthesis with precise sequence control.
• To integrate electronically controlled deprotection chemistry (ECDC) with an on-chip hydrogel-primer system for automated DNA synthesis.
• To demonstrate the feasibility and accuracy of the developed platform for single-stranded DNA (ssDNA) production.

Main Methods:

• Development of an on-chip system combining ECDC with a hydrogel-primer modification strategy.
• Utilizing electrochemically generated nitrous acid (HNO2) for selective deprotection of 3'-oxyamino groups on DNA primers.
• Implementing a multipixel synthesis array on silicon chips for parallel DNA synthesis with single-base resolution.

Main Results:

• The ECDC platform enabled precise spatiotemporal control over the enzymatic DNA synthesis process.
• Validation experiments demonstrated 100% accuracy for single-sequence synthesis and an average of 96% accuracy for dual-sequence synthesis.
• The system achieved parallel synthesis of ssDNA with single-base resolution on silicon chips.

Conclusions:

• The novel ECDC-integrated platform offers a highly accurate solution for high-throughput ssDNA synthesis.
• This technology lays the foundation for scalable and automated enzymatic DNA manufacturing.
• The developed system advances the field of next-generation DNA synthesis technologies.