Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing

Area of Science:

• Molecular Biology
• Biotechnology
• Nanotechnology

Background:

• Rolling circle amplification (RCA) using phi29 DNA polymerase (Polφ29) synthesizes long DNA strands from circular templates.
• Single-stranded circular DNA templates (ssCDTs) limit RCA's DNA synthesis rate.
• Faster DNA synthesis is needed for advanced molecular detection applications.

Purpose of the Study:

• To overcome the slow DNA synthesis rate associated with ssCDTs in RCA.
• To engineer a novel DNA template for enhanced Polφ29 activity.
• To develop a rapid and sensitive microRNA detection platform.

Main Methods:

• Engineered a micrometer-sized DNA track (μDT) template: a long linear ssDNA with repeating units and a primer-binding domain.
• Utilized phi29 DNA polymerase (Polφ29) for DNA synthesis on the μDT template.
• Designed and implemented an all-in-one printed paper device for microRNA detection.

Main Results:

• Polφ29 exhibited a ≈5-fold faster DNA synthesis rate with the μDT template compared to ssCDTs.
• The μDT template facilitated efficient rolling circle amplification.
• The paper-based sensor detected 1 pM let-7a microRNA within 10 minutes.

Conclusions:

• Micrometer-sized DNA tracks (μDTs) significantly enhance Polφ29 DNA synthesis speed.
• This μDT-based RCA approach enables rapid and sensitive microRNA detection.
• The developed paper sensor offers a simple, efficient platform for point-of-care diagnostics.