Computing in mammalian cells with nucleic acid strand exchange.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• DNA strand displacement is established for cell-free molecular systems.
• In-cell applications of DNA strand displacement are limited.
• Interfacing DNA circuits with native cellular components like mRNA is challenging.

Purpose of the Study:

• To adapt DNA strand displacement and exchange reactions for operation within mammalian cells.
• To engineer DNA circuitry capable of directly interacting with native messenger RNA (mRNA).
• To demonstrate the feasibility of complex logic operations and gene regulation using in-cell DNA circuits.

Main Methods:

• Optimized fluorescent reporters based on four-way DNA strand exchange reactions.
• Systematically varied molecular structure, chemistry, and delivery methods for cellular performance.
• Developed and tested AND/OR logic gates using four-way strand exchange.
• Activated small interfering RNA (siRNA) via strand exchange reactions.
• Utilized native mRNA as scaffolds for co-localizing DNA logic gates.

Main Results:

• Identified robust design principles for in-cell DNA circuitry performance.
• Demonstrated multi-input logic gates (AND, OR) operating within mammalian cells.
• Successfully activated functional siRNA through DNA strand exchange.
• Achieved subcellular resolution visualization of DNA circuitry operation using mRNA scaffolds.

Conclusions:

• DNA strand displacement circuitry can be effectively implemented in mammalian cells.
• Native mRNA can serve as a programmable scaffold for in-cell DNA logic.
• This technology offers a new platform for sophisticated molecular programming and gene regulation in biological environments.