Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids.

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Cell-free protein synthesis (CFPS) offers a platform for nucleic acid detection.
• Existing CFPS systems often utilize toehold switches but suffer from leaky translation, high detection limits (~30 nM), and laborious screening.
• These limitations hinder the efficiency and accuracy of nucleic acid detection using CFPS.

Purpose of the Study:

• To develop an improved CFPS system for nucleic acid detection that overcomes the limitations of toehold switch systems.
• To create a highly sensitive, specific, and user-friendly platform for detecting target nucleic acids.
• To establish a versatile core platform for diverse nucleic acid detection applications.

Main Methods:

• Development of a split T7 switch-mediated CFPS system utilizing a three-way junction structure for selective transcription-translation initiation.
• Construction of fluorescence and colorimetric detection systems using different reporter proteins.
• Incorporation of self-complementation of split fluorescent proteins for streamlined system preparation.
• Application of a one-pot, isothermal approach for nucleic acid detection.

Main Results:

• The split T7 switch system enables transcription-translation initiation exclusively in the presence of target nucleic acids, minimizing off-target signals.
• Achieved a detection limit as low as 10 pM for target nucleic acids, representing over a thousand-fold improvement compared to previous toehold switch methods.
• Demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins.
• The system streamlines preparation and enables versatile applications through self-complementation of split fluorescent proteins.

Conclusions:

• The developed split T7 switch-mediated CFPS system effectively overcomes critical limitations of existing toehold switch-based methods.
• This novel system provides a significant advancement in sensitivity and specificity for nucleic acid detection.
• The platform's versatility, ease of use, and improved performance position it as a core technology for future nucleic acid detection applications.