Precise, Orthogonal Remote-Control of Cell-Free Systems Using Photocaged Nucleic Acids

Area of Science:

• Synthetic Biology
• Biotechnology
• Molecular Biology

Background:

• Cell-free gene expression is crucial for nanotechnology and synthetic biology.
• Precise, noninvasive control of cell-free systems using light is highly desirable but challenging.
• Development of light-controlled OFF switches for cell-free expression has lagged behind ON switches.

Purpose of the Study:

• To develop orthogonally light-controlled cell-free expression OFF switches.
• To enable precise, remote, and noninvasive modulation of gene expression in cell-free systems.
• To integrate light-controlled OFF switches with existing ON switches for dynamic control.

Main Methods:

• Attaching nitrobenzyl and coumarin photocages to antisense oligonucleotides to create light-controlled OFF switches.
• Utilizing commercially available oligonucleotides for switch synthesis.
• Demonstrating orthogonal mRNA degradation using different light wavelengths.
• Combining developed OFF switches with a previously established blue-light-activated DNA template ON switch.

Main Results:

• Successfully developed orthogonally light-controlled cell-free expression OFF switches.
• Achieved tight control over cell-free expression.
• Demonstrated wavelength-dependent orthogonal degradation of two distinct mRNAs.
• Showcased sequential control of transcription (ON) and translation (OFF) using different light wavelengths.

Conclusions:

• The developed photocaged antisense oligonucleotides provide effective, orthogonal light-controlled OFF switches for cell-free expression.
• This technology enables precise, remote, and dynamic ON/OFF control of gene expression in cell-free systems.
• The advancement is significant for applications in synthetic biology, biological logic gates, and synthetic cells.