Collaboration
Introduction
The Lethbridge High School team is developing a multifunctional mRNA construct to treat type 1 diabetes in project Dia-Beatable. They are developing self-amplifying and self-regulating sa-mRNA molecules to control insulin production. Through this project, the team aims to provide a novel and effective treatment for type 1 diabetes patients.
The UManitoba team is helping the Lethbridge High School team to test and validate the glucose sensitivity of the mRNA construct. To achieve glucose sensitivity, a riboswitch that can bind to glucose is incorporated into the mRNA. Upon binding to glucose, this riboswitch can adopt a suitable conformation to regulate the mRNA construct and trigger insulin expression.
Riboswitches can be tested by fusing them with fluorogenic aptamer, which can then adopt a suitable conformation to bind to a pro-fluorescent dye. A complex of pro-fluorescent dye and fluorogenic aptamer can fluoresce and thus, enable qualification. Hence, a fusing construct is created by fusing glucose-sensing riboswitch with RNA Mango, a fluorogenic aptamer. Upon binding to glucose, the riboswitch can trigger RNA Mango to adopt a suitable conformation to bind to a pro-fluorescent dye called TOI, which enables detection using fluorescent spectroscopy.
The Lethbridge High School team sent their glucose sensing aptamers construct and TOI dye to the UManitoba team. The UManitoba team prepared TAKEM4 buffer, glucose solution, and nuclease-free water for the experiment. The UManitoba team then performed five experiments designed by the Lethbridge High School team, three of which are reported below.
TOI Background Fluorescence
Figure 1. Fluorescent of TOI dye in absence of RNA biosensor and increasing glucose concentration. Samples are mixed and transferred to a quartz cuvette for fluorescent measurement. Measurements are done using γ Excited = 510 nm and γ Emission = 520 – 600 nm.
Glucose Sensitivity of Scramble Aptamer
Figure 2. Glucose sensitivity of scramble aptamer. Samples are mixed and transferred to a quartz cuvette for fluorescent measurement. Measurements are done using γ Excited = 510 nm and γ Emission = 520 – 600 nm.
Glucose Sensitivity of Glucose Aptamer
Figure 3. Glucose sensitivity of glucose aptamer. Samples are mixed and transferred to a quartz cuvette for fluorescent measurement. Measurements are done using γ Excited = 510 nm and γ Emission = 520 – 600 nm.
Contribution to Lethbridge_HS
Confirmation: the presence of glucose leads to an increase in fluorescence, the intended function of the aptamers.
Novel Observation: Incubating the sample for a longer period of time with glucose leads to higher fluorescence. The results shared with Lethbridge, they will draw the final conclusion. In our opinion it takes about 5 min to reach the equilibrium conditons.
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