Topologically switchable and gated transcription machinery.

Area of Science:

• Synthetic biology
• Molecular engineering
• Biochemistry

Background:

• Topological barriers naturally regulate gene expression by controlling transcription machinery.
• Developing synthetic systems to mimic and control these natural processes is crucial for advancing biotechnology.

Purpose of the Study:

• To design synthetic DNA templates with switchable topological barriers for triggered RNA aptamer transcription.
• To demonstrate controlled and reversible transcription of specific RNA aptamers using these novel templates.

Main Methods:

• Utilized Sr²⁺-ion-stabilized G-quadruplex and kryptofix [2.2.2] (KP) as reversible topological barriers.
• Employed T-A·T triplex barriers separated by fuel strands for cyclic transcription.
• Incorporated photoactivated azobenzene-modified nucleic acid barriers for "ON"/"OFF" transcription control.
• Integrated DNA tetrahedra scaffolds and T7 RNA polymerase within hydrogel microcapsules for cell-like assemblies.

Main Results:

• Demonstrated switchable transcription of malachite green (MG) RNA aptamer using G-quadruplex/KP barriers.
• Achieved cyclic triggered transcription of a DFHBI-binding aptamer using T-A·T triplex barriers.
• Showcased "ON"/"OFF" control of MG aptamer transcription with photoresponsive barriers.
• Successfully gated transcription of MG or DFHBI aptamers using a mixture of topological triggers.
• Established reversible transcription of MG aptamer within DNA hydrogel microcapsules.

Conclusions:

• Developed versatile synthetic DNA topological barriers for precise control over RNA aptamer transcription.
• These systems offer a powerful platform for switchable and triggered gene expression.
• The cell-like microcapsule assemblies demonstrate potential for in vitro synthetic biology applications.