Deaminase-based RNA Sensors

Technology

The inventors have created an RNA-editing sensor system termed RADAR that can express an effector protein in the presence of a target RNA species. The sensor system comprises a single-stranded RNA (ssRNA) sensor and an adenosine deaminase acting on RNA (ADAR) deaminase. The ssRNA sensor consists of a stop codon and a payload and is capable of binding to a ssRNA target to form a dsRNA substrate. 

The method includes creating a dsRNA region via hybridization of the ssRNA sensor to an endogenous transcript, which generates an A-C mismatch within the dsRNA substrate. Upon binding to the endogenous target transcript by the ssRNA sensor, the stop codon (UAG) on the sensor is changed to a sense codon (UIG) by ADAR, which enables translation and allows the payload to be expressed. 

Problem Addressed

Due to advances in single-cell technologies, there are a large number of cell types and states that can now be measured and defined by expressed RNA. However, there is still a lack of strictly observational sensor systems to track and measure RNA levels in vivo. Additionally, techniques that integrate sensors to track RNA levels require the use of transgenic organisms, which are often not feasible in many scenarios due to their imprecision and inflexibility. A strictly observational RNA sensor would enable cell-state specific tracking and perturbation with high sensitivity, thereby overcoming the unforeseen consequences on gene expression inherent with current methods. 

Advantages

• RADARs can function with either endogenous cellular ADAR or exogenous ADAR.  

• Ability to control expression of synthetic mRNA (important for mRNA therapeutics/vaccines). 

• Detection sensitivity on endogenous transcripts as low as 13 transcripts per million (TPM). 

• Various payloads can be attached to the sensor system (e.g., luciferase, fluorescent proteins, and caspases).  

• Expressed via DNA or synthetic mRNA. 

Publications

Kaiyi Jiang, Jeremy Koob, Xi Dawn Chen, Rohan N. Krajeski, et al. Programmable Eukaryotic Protein Expression with RNA Sensors. BioRxiv 2022 jan 26. doi: 10.1101/2022.01.26.477951