The goal of this project is to develop RNA fluorescent stickers that can sense fever. This helps to prevent the spread of infectious diseases that involves fever by detecting patients in a relatively easy manner. Our team combined the CHA method and thermosensitive RNA to introduce a system that emits fluorescence above a specific temperature and incorporated that system into stickers.
Considering functionality and comfort, we suggest the following design of the sticker. 'Thermopatch' is divided into a adhesive part which attaches to the skin and the capsule part that contains RNA and buffer. The capsule is further divided into two subcapsules named the dry capsule and the wet capsule. The dry capsule contains RNA in a dried form that is optimized for long-term storage and the wet capsule contains the buffer that rehydrates and activates the RNA upon usage. When the user presses the wet capsule, the buffer is released and the system is activated by the rehydration of RNA.
- RNA is single stranded so it is a very fragile molecule. It is easily degraded by RNase. As a result, it cannot be stored at room temperature for a long time. Therefore, we came up with an idea to store RNA in a dry state using Biomatrica's RNAstable technology. In a dry state, RNases are not able to act on the RNA so RNA stability is ensured. Biomatrica's RNAstable products have been proven to be stable for up to 3.5 years at room temperature. Moreover, upon rehydration, the stored RNA will recover 100% within 10 minutes and can be used immediately without any further purification process.
- We confirmed in paper, "Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells", that the CHA system works most efficiently in a buffer of pH7.5, containing 10mM Tris, 5mM MgCl2, 100mM KCl, and 10mM NaCl. Especially, It is known that magnesium concentration plays an important role in the efficiency of CHA. Maximum fluorescence intensity of CHA was enhanced at higher magnesium concentrations. However, the background signal from leakage also increased. Thus, optimal signal-to-background ratio was observed at magnesium concentration between 1 and 5mM. Indeed, CHA can perform well with physiological concentrations of magnesium ions.
genetically encoded light-up RNA aptamers
Reference : Ouellet, J. (2016). RNA fluorescence with light-up aptamers. Frontiers in chemistry, 4, 29.
Aptamer is a nucleic acid capable of strongly binding to a particular molecule while maintaining a stable tertiary structure. Genetically encoded light-up RNA aptamers, in particular, refer to RNA sequences that can bind with their cognate nonfluorescent fluorogens and greatly activate their fluorescence. It is new tools with high sensitivity and robustness for intracellular detection.
Theoretically, it is always possible to design RNA suitable for a certain fluorogen. Compared to fluorescent protein based sensors, light-up RNA aptamer based sensors are of easier programmability, which allows the detection of small ligands and native RNAs. Genetically encoded light-up RNA aptamer based sensors generally possess high signal-to-background ratios, excellent specificity and fast kinetics. Especially, it shows high stability in cell free system.
Several types of aptamers have been developed for each fluorogen. We chose Broccoli among them. The following shows typical types of fluorophores and their excitation and emission properties.
reference: Swetha, P., Fan, Z., Wang, F., & Jiang, J. H. (2020). Genetically encoded light-up RNA aptamers and their applications for imaging and biosensing. Journal of Materials Chemistry B, 8(16), 3382-3392.
Broccoli is an aptamer that binds to DFHBI and emits fluorescence. Broccoli is an improved version of Spinach, which shows high folding efficiency similar to Spinach2, but has a low dependence on Mg2+ for folding and higher thermal stability. This allowed a high percentage of correct folding and enhanced fluorescence.
What is Catalytic Hairpin Assembly?
The RNA CHA method is a highly efficient isothermal amplification method which does not require enzymes. This method provides higher catalyst efficiency, lower background signals, and a simpler and more stable response system, so it is suitable for detecting fever.
This method can be described simply in the figure below.
Design of CHA
1. Broccoli was separated into two nonfluorescent parts, termed Broc and Coli. -> low fluorescence signal
2. Fused Broc to the 3' end of the H1 hairpin and Coli to the 5' end of the H2 hairpin.
3. The active fluorogenic form of Broccoli can only be formed upon target-driven H1 and H2 hybridization.
modus operandi of CHA
Broccoli was separated into two nonfluorescent parts, termed Broc and Coli, which was conjugated to the end of two CHA hairpins, H1 and H2, respectively. Spontaneous hybridization of two CHA hairpins is kinetically hindered by embedding the complementary regions within the hairpin stems. Only in the presence of a target input strand(C) which can change their structure depending on temperature, H1 hairpins can be opened and will further enable the assembly of both hairpins. Only when they are reunited into the original Broccoli structure, the fluorescence signal of DFHBI-1T can be recovered. Eventually the target strand can be spontaneously displaced and recycled, similar to a catalyst, to induce more hairpin assembly events.
reference: Karunanayake Mudiyanselage, A. P., Yu, Q., Leon-Duque, M. A., Zhao, B., Wu, R., & You, M. (2018). Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells. Journal of the American Chemical Society, 140(28), 8739-8745.
As shown in the graph above, the system shows optimal in vitro CHA assembly efficiency around 22°C. We will move optimal activity to 37.5 degrees by increasing the base pairs of C to apply this to body temperature detection.
Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells, Aruni P K K Karunanayake Mudiyanselage et al., J Am Chem Soc, 2018.