RNA S enSor based Analysis system in Yeast IMPORTANT THINGS
RNA is a lonely traveler through space and time.
Throughout the journey, RNA has established its important position.
The Problem
Developing molecular sensing tools at the RNA level to monitor gene expression in space and time helps understand the complex regulatory mechanisms within cells and is fundamental for the design and control of engineered life systems. And tremendous efforts have been made to uncover the mystery of RNA. Available RNA detection methods include qPCR, RNA-seq, fluorescence in situ hybridization (FISH), and type III-E CRISPR all have their own disadvantages.
It seems impossible to combine the advantages of in vivo detection, large detection numbers, high spatial resolution, low cost of property and manpower, and high expandability.
But is it real?
Click to know more about available RNA detection methods' disadvantages
| Transcription Level Detection Method | Environment | RNA Detection Numbers | Spatial Resolution | Cost | Expandability |
|---|---|---|---|---|---|
| qPCR | In vitro | Small | Not In situ | Low | Low |
| RNA seq | In vitro | Large | Not In situ | High | Low |
| FISH | In vivo | Large | In situ | High | Low |
Table. Available RNA detection methods' disadvantages
Experience tells us one can not easily get along with RNA, due to its fragile structure and complicated functions. But fortunately, there has been its natural friend just inside our body——ADAR.
ADAR, adenosine deaminase acting on RNA, can specifically recognize A-C mismatched base pairs on double-stranded RNA and achieve high-precision A to I (adenine to inosine) editing.
Our Solution
Using this feature, artificial RNA can be designed to target any endogenous RNA in cells and form hybrid double-stranded RNA regions. By introducing a UAG stop codon on the artificial RNA and an ACC codon on the endogenous RNA to form a single base mismatch, ADAR enzymes are recruited to initiate their editing activity.
The resulting UIG codon is usually recognized by the ribosome as UGG (non-stop codon), thereby initiating the translation of downstream transcripts. And using downstream transcripts that produce a signal we could detect will show information of the target RNA we want to know.
Utilizing this concept, highly programmable RNA sensing and regulation can be achieved.
Visulization of Our Solution
Our experimental principle demonstration video is presented below.
Click to know more about some core results of our experiments
Pic. Confocal Results of Editing Systems Containing Different MS2 sequences
Pic. Confocol Results of Confocal Results of Sensor-target_Chk1(s)-Chk1(s)
Pic. FACs Results of Editing Systems Containing Different MS2 sequences
Pic. FACs Results of Sensor-target_Chk1(s)-Chk1(s)
Application
Detect multigene expression
Strain Security System
Monitor splice variants
In situ detection of multigene expression
The very basic application of our system is to carry out In situ detection of multigene translation. By applying different sensors for different target, we can read the target RNA level from fluorescence intensity in real time. One possible application scenarios could be to apply our system to the production of liquor. By reading the RNA level in Saccharomyces cerevisiae for specific enzymes, enterprise managers can have better predict and regulate the flavor of liquor.
Engineering Strain Security System
Engineering strains are the "chips" of bio-manufacturing, and protecting their genetic information is crucial. In China, since 2017, there has been many microbial invention patent infringement disputes, bring attentions to the field of strain security. We used our RNA sensor to construct a strain security system: the protected strain will only be thawed successfully under special environmental stress like starvation or high temperature, thus stopping the strain stealing in the microbial strain repository
Application for in vivo dynamic monitoring of splice variants
The ratio between splice variants are important signals for the cellular state, including cancerization. But no user-friendly molecular tool to detect the ratio has been developed. The RNA sensor we developed possess the potential to distinguish splice variants and uncover the secret hidden behind the ratio of them.