TY - JOUR
T1 - Generation of small molecule-binding RNA arrays and their application to fluorogen-binding RNA aptamers
AU - Henderson, Charlotte
AU - Rail, Callum
AU - Butt, Louise
AU - Vincent, Helen
AU - Callaghan, Anastasia
PY - 2019/9/1
Y1 - 2019/9/1
N2 - The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.
AB - The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.
KW - RCUK
KW - BBSRC
KW - BB/I532988/1
KW - BB/L017628/1
UR - https://www.sciencedirect.com/science/article/pii/S104620231830375X?via%3Dihub
U2 - 10.1016/j.ymeth.2019.04.021
DO - 10.1016/j.ymeth.2019.04.021
M3 - Article
SN - 1046-2023
VL - 167
SP - 39
EP - 53
JO - Methods
JF - Methods
ER -