Programmable AND-gate strategy to reduce false positives in rolling circle amplification

Improving the specificity of biosensors by reducing their false positive rate is a long-standing challenge, particularly for point-of-care (POC) diagnostics. An emerging and potentially impactful class of POC biosensor is those that can detect specific nucleic acid sequences. These sensors often use isothermal amplification of nucleic acid sequences, which is considered suitable for POC use due to its relative simplicity and its lack of requirement for specialized equipment or highly trained personnel. However, isothermal amplification methods are still quite susceptible to false positives, especially those like Rolling Circle Amplification (RCA) that amplify nucleic acids under mild temperature conditions that are more prone to near-target amplification. To address this limitation, we introduced an AND-gate Boolean logic to create Multi-Key RCA, which uses detection of multiple nucleic acid sequences as a prerequisite to amplification. Multi-Key RCA requiring two DNA sequences not only markedly reduced false positives compared to conventional RCA but also achieved high specificity in single-nucleotide polymorphism (SNP) detection. We showed that Multi-Key RCA also can detect RNA and can be expanded to use AND gates with more than two inputs. We integrated Multi-Key RCA with a lateral flow assay to create a user-friendly synthetic biology-based diagnostic platform requiring no specialized equipment, and we showed that this diagnostic can be implemented as a one-pot reaction that integrates all RCA steps and can be lyophilized for eventual long-term storage stability. Overall, this Boolean logic-based synthetic biology approach shows significant promise to reduce false positives in field-deployable nucleic acid biosensors.