Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biosensor Engineering

Background:

• Toehold switches are programmable RNA biosensors for detecting viral RNAs.
• Current designs suffer from leaky translation, reducing sensitivity and fold change.
• Engineering new toehold switches is time-consuming and unpredictable.

Purpose of the Study:

• To engineer a signal amplification circuit to improve toehold switch performance.
• To address leaky translation and enhance the fold change and sensitivity of toehold switches.
• To create a broadly applicable circuit for biosensor applications.

Main Methods:

• Constructed and tested signal amplification circuits using serine integrase.
• Evaluated circuits with toehold switches targeting Dengue virus RNA, SARS-CoV-2 RNA, and an artificial RNA.
• Quantified improvements in fold change and detection limits.

Main Results:

• The serine integrase circuit effectively suppressed leaky translation in the OFF state.
• Expression fold change was significantly boosted from OFF to ON states.
• Detection limits were reduced by 3-4 orders of magnitude for targeted RNAs.
• The circuit converted analog biosensor signals into digital-like outputs.

Conclusions:

• The serine integrase circuit is a broadly applicable solution for enhancing toehold switch biosensors.
• This approach overcomes limitations of leaky translation and improves sensitivity.
• The circuit simplifies the process of developing high-performance RNA biosensors.