Poster Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

3-bit fluorescence reporting using RNA aptamers (#13)

Bradley I. Harding 1 , Shelley F.J. Wickham 1 2 3
  1. School of Chemistry, University of Sydney, Sydney, NSW, Australia
  2. School of Physics, University of Sydney, Sydney, NSW, Australia
  3. The University of Sydney Nano Institute, University of Sydney, Sydney, NSW, Australia

Multibit reporting is important to understand the output of DNA-based computational or diagnostic reactions. Multiplexing, the combination of multiple signals into a single, complex signal can achieve multibit reporting. Commonly used systems include colour multiplexing with multiple fluorophores and spatial multiplexing in multi-pot reactions. More recently, temporal multiplexing, such as qPAINT,1 and temperature modulation2 methods have been developed. However, these are typically limited to four to six bits per sample depending on instrument and probes used. In this work we have developed a 3-bit reporter using amplitude modulation, based on programmable inhibition of a fluorogenic RNA aptamer in an enzyme-free system.


Fluorogenic RNA aptamers, such as Spinach and Broccoli, light-up on interaction with the small molecule DFHBI3 and can be used as reporters for in vivo and in vitro transcriptional circuits.4–6 Fluorescence switch on occurs when polymerases are activated to transcribe the RNA aptamer in the presence of DFHBI. Here we take a different approach by inhibiting the interaction of DFHBI and the aptamer rather than inhibiting the aptamer transcription rate. This was achieved be designing 3 short ssDNA inhibitors that are partially complementary to the aptamer and have unique toehold sequences. Inhibitors are combined with the aptamer at a fixed ratio of concentrations of 4:2:1. Input of an activator strand complementary to only 1 inhibitor activates only a sub-population of the aptamer resulting in a well-defined fluorescence intensity. Different patterns of activators result in different fluorescence intensities. We tested the robustness of the reporter through multiple rounds of cycling OFF and ON and demonstrated transitioning from one input signal to another. The system recovered full fluorescence intensity after three cycles of 90 minutes OFF/ON. When compared to in vitro transcriptional circuits this reporter system does not require enzymatic conditions, responds faster to the input, is resettable, and can incorporate more information. Overall, this method achieves fluorescence reporting with amplitude multiplexing. Limitations include that the RNA can be sensitive to degradation and that activators and inhibitors must be correctly normalised.

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