Chapter 5
Development and Applications of Fluorogen/Light-Up RNA
Aptamer Pairs for RNA Detection and More
Michael Ryckelynck
Abstract
The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive
technologies allowing for imaging the synthesis and the location of these molecules in living cells. This
need motivated the development of small pro-fluorescent molecules called “fluorogens” that become
fluorescent upon binding to genetically encodable RNAs called “light-up aptamers.” Yet, the development
of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the
fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs.
Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is
presented and discussed.
Key words Fluorogen, Aptamer, RNA, Functional screening, SELEX, Live-cell imaging, Biosensing,
Engineering
1 Introduction
RNA is a main actor of cell life mainly through its central role in
gene expression and its regulation. Indeed, RNA has pleiotropic
functions such as being the message (messenger RNA or mRNA) to
be translated into protein, and the regulator of gene transcription,
mRNA maturation, and/or translation (e.g., noncoding regulatory
RNAs), as well as being the active component of key cellular
machineries (e.g., ribosomal RNA, small nuclear RNAs, and
RNase P). It is therefore of prime importance to be able to monitor
the expression and ideally also the location of RNAs all along the
cellular life span. Yet, for a long time, the detection of cellular
nucleic acids was restricted to the use of nonspecific intercalating
dyes (e.g., Hoechst [1] and cyanines [2]) and to in situ hybridization (ISH) methodologies [3], in which radioactively or fluorescently labeled oligonucleotides (DNA or RNA) are used to detect
RNA molecules upon specific annealing of the probe. Whereas ISH
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020
73
Development and Applications of Fluorogen/Light-Up RNA
Aptamer Pairs for RNA Detection and More
Michael Ryckelynck
Abstract
The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive
technologies allowing for imaging the synthesis and the location of these molecules in living cells. This
need motivated the development of small pro-fluorescent molecules called “fluorogens” that become
fluorescent upon binding to genetically encodable RNAs called “light-up aptamers.” Yet, the development
of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the
fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs.
Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is
presented and discussed.
Key words Fluorogen, Aptamer, RNA, Functional screening, SELEX, Live-cell imaging, Biosensing,
Engineering
1 Introduction
RNA is a main actor of cell life mainly through its central role in
gene expression and its regulation. Indeed, RNA has pleiotropic
functions such as being the message (messenger RNA or mRNA) to
be translated into protein, and the regulator of gene transcription,
mRNA maturation, and/or translation (e.g., noncoding regulatory
RNAs), as well as being the active component of key cellular
machineries (e.g., ribosomal RNA, small nuclear RNAs, and
RNase P). It is therefore of prime importance to be able to monitor
the expression and ideally also the location of RNAs all along the
cellular life span. Yet, for a long time, the detection of cellular
nucleic acids was restricted to the use of nonspecific intercalating
dyes (e.g., Hoechst [1] and cyanines [2]) and to in situ hybridization (ISH) methodologies [3], in which radioactively or fluorescently labeled oligonucleotides (DNA or RNA) are used to detect
RNA molecules upon specific annealing of the probe. Whereas ISH
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020
73