Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

Abstract

The central role of RNA in living systems made it highly desirable to have non-invasive 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-e ce ec e ca ed e a bec e e ce b d e e ca e c dab e RNA ca ed-a a e. Ye , e 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 will be presented and discussed.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field

“…Various aptamers now can be routinely engineered through repeated rounds of in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX), the powerful technique that was developed in the early 1990s. , Since then, numerous different types of aptamers against different target molecules have been selected in academic research laboratories and have found applications in various biotechnological fields. − In particular, the fluorogenic RNA aptamers are known for their specificity in binding otherwise nonfluorescent dyes; the fluorescent emission of the dye is activated only upon binding to its RNA aptamer. This binding specificity enabled the design not only of label-free nucleic-acid-based sensors in vitro − but also the attractive development of binary 1 and 0 systems, as shown in Figure D,E. , The input signal induces the conformational change of the fluorophore-binding pocket (analogous to a “gate”), dictating the overall affinity strength of the RNA–fluorophore complex. Various factors can potentially serve as inputs, including single-stranded oligonucleotides and nonfluorescent molecules mimicking structures of the ligand dye.…”

Structure-Informed Design of an Ultra Bright RNA-activated Fluorophore

“…17,19,20,21 The Mango II aptamer contains a well-de ned but plastic pocket capable of binding TO derivatives in multiple orientations. To date, most work on the Mango system has focused on evolution of improved aptamers, 22,23 development of uorogenic transcripts for live cell or single molecule imaging, 24 or rational modi cation of the TO uorophore to alter excitation/emission pro les. 25 The discovery of Mango aptamers represents a powerful advance in the development of RNA imaging tools.…”

Rapid and Multiplexed Nucleic Acid Detection using Programmable Aptamer-Based RNA Switches

“…Fluorescent light-up aptamers have emerged as powerful tools for visualizing the dynamics of RNA expression in living cells [1][2][3] and providing a bright, translation-free readout for in vitro reactions [4][5][6] . These systems consist of short sequences of RNA or DNA that bind to conditionally fluorescent dye molecules known as fluorogens to activate their fluorescence.…”

“…These systems consist of short sequences of RNA or DNA that bind to conditionally fluorescent dye molecules known as fluorogens to activate their fluorescence. Since the 2003 discovery of the MG RNA aptamer for binding malachite green 7 , diverse aptamer/fluorogen pairs have been developed with high binding specificity and varying spectral and photophysical properties 1 . In 2014, Jaffrey et al reported the Broccoli aptamer that binds to the GFP-mimicking fluorogen DFHBI-1T and provides robust folding and green fluorescence in living cells 8 .…”