Generalized Strategy for Engineering Mammalian Cell-Compatible RNA-Based Biosensors from Random Sequence Libraries
Year: 2023
Type: article
Abstract: Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by Capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.
Source: ACS Sensors
Institution: Vanderbilt University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Vanderbilt University, Chan Zuckerberg Initiative, Georgia Clinical and Translational Science Alliance, National Institutes of Health, Vanderbilt Digestive Diseases Research Center, Vanderbilt University Medical Center, National Eye Institute, National Cancer Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Center for Research Resources, National Center for Advancing Translational Sciences
Awards: G1344704808, G153582612, G1800433781, G1812229208, G1845105935, G2043477120, G2268269956, G2615776415, G3051419658, G3433274139, G4020890471, G4512720654, G5580019600, G5591816454, G563474107, G577112812, G5853943267, G659731888, G7220527126, G725763919, G7520984233, VANTAGE:Consolidation to create the Vanderbilt Technologies for Advanced Genomics, G7642566612, G834632907, Molecular and Cellular Basis for Digestive Diseases, Vanderbilt Institute for Clinical and Translational Research (VICTR), Core Grant in Vision Research, G949362299, G95160140
OpenAlex ID: W4375853379