Application of real-time RNA-based sensors to study concentrations of metabolites and other physiological events by fluorescence is hindered by inadequate expression of RNA by mammalian cells. We have previously developed RNA-based sensors by combining the dye- activating fluorogenic RNA aptamer, Spinach, with metabolite-binding RNA riboswitch elements and implemented them in E. coli. When expressed in mammalian cells, such RNA-based sensors are not as abundant as in E. coli and lack any fluorescence signal. Recent findings have indicated that metazoan tRNA introns generate stable circular RNAs (circRNA) in vivo that can be detected by fluorescence microscopy when incorporating a Spinach-like aptamer sequence. CircRNAs exhibit longer half-lives than their corresponding linear RNAs, possibly due to their resistance to degradation by endogenous exoribonucleases; however in vivo expression of circRNAs is low. Preliminarily, we have devised a new approach to generating circRNAs endogenously that demonstrates 20-fold higher expression levels than the tRNA-intron-based approach. This technology presents an opportunity to express circRNA-based sensors in mammalian culture for the first time and for a range of applications when combined with existing RNA biotechnology. On this basis, I propose to optimize this circRNA expressing technology for increased concentration to improve the fluorescence signal of circRNA-based sensors and expand overall usefulness. Furthermore, I intend to adapt our approach to linear RNA-based sensors to a circRNA context, considering the associated structural and sequence constraints. Finally, I will design the first circRNA-based sensor in mammalian cells by incorporating previously identified RNA aptamers against the truncated polymerase of the hepatitis B virus (HBV). We will also improve on previous approaches to sensor design by optimizing critical transducer sequences of the sensor in a high-throughput manner using next-generation sequencing. Such an optimized sensor will be the basis for a novel HBV infection reporter that does not require engineering of the HBV genome in ways that alter its normal life cycle, as has been done for 25 years. An HBV reporter system that does not intrinsically change the virus?s replication and infection behavior will allow fundamental discoveries as to the mechanism of HBV pathogenesis.
Developing fluorescent reporters hepatitis B virus (HBV) infection is especially difficult ? current methods that engineer the HBV genome perturb the normal viral life cycle because the HBV genome is small and tightly regulated by cis-elements. I aim to improve upon technology for stable RNA expression, enabling modular and rapid implementation of fluorescent RNA-based biosensors for physiological events and other applications in RNA biotechnology. Achieving high expression of stable RNA will allow me to create a non-perturbative HBV infection reporter that enables breakthroughs in the fundamental aspects of the mechanisms of HBV pathogenesis and new approaches to therapeutics discovery.
