Light-Up Split Broccoli Aptamer as a Versatile Tool for RNA Assembly Monitoring in Cell-Free TX-TL Systems, Hybrid RNA/DNA Origami Tagging and DNA Biosensing

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a 'bio-orthogonal' hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets.

Automated summary

Binary light-up aptamers are versatile tools for various applications. We demonstrated the functionality of a split Broccoli aptamer system that only emits fluorescence in the presence of a complementary sequence. The system was successfully assembled in an E. coli-based cell-free TX-TL system and a hybrid RNA/DNA rectangle origami, showing the effectiveness of the split system. The system was also used to detect a Campylobacter spp. DNA target sequence, highlighting its potential applications in real-time monitoring and detection of nucleic-acid-based devices.