Structure-based insights into fluorogenic RNA aptamers

Abstract

Fluorogenic RNA aptamers are in vitro-selected RNA molecules capable of binding to specific fluorophores, significantly increasing their intrinsic fluorescence. Over the past decade, the color palette of fluorescent RNA aptamers has greatly expanded. The emergence and development of these fluorogenic RNA aptamers has introduced a powerful approach for visualizing RNA localization and transport with high spatiotemporal resolution in live cells. To date, a variety of tertiary structures of fluorogenic RNA aptamers have been determined using X-ray crystallography or NMR spectroscopy. Many of these fluorogenic RNA aptamers feature base quadruples or base triples in their fluorophore-binding sites. This review summarizes the structure-based investigations of fluorogenic RNA aptamers, with a focus on their overall folds, ligand-binding pockets and fluorescence activation mechanisms. Additionally, the exploration of how structures guide rational optimization to enhance RNA visualization techniques is discussed.

1. Introduction

RNA is one of the most fundamental biomacromolecules in life and plays a crucial role in diverse biological processes, such as genetic information translation, gene expression regulation, and maintenance of cell functionality [1‒4]. The visualization of RNA localization and dynamics with high spatial and temporal resolution is essential for investigating their functions, mechanisms and interactions in biology. Fluorescent proteins, especially green fluorescent protein (GFP), have revolutionized the spatiotemporal localization of proteins and the investigation of protein interactions both in vivo and in vitro [5‒8]. However, intrinsic fluorescent RNAs comparable to GFP have not been identified until now. Current techniques used for the dynamic detection of RNA molecules include fluorescence in situ hybridization (FISH) [9,10], molecular behavior technology [11], and RNA hairpin methods [12‒18]. While effective, FISH requires cell fixation and cannot be used for live-cell imaging [9,10]. Molecular behavior technology allows spatiotemporal imaging of RNA molecules in living cells but is limited by false-positive signals [11]. Alternatively, RNAs of interest can be labelled with naturally occurring RNA hairpins along with their specific binding proteins (e.g., MS2-MCP [12], PP7-PCP [14], λN22-boxB [15] and gRNA-dCas [16‒18]) fused to fluorescent proteins. Unfortunately, this technique suffers from the high background fluorescence of unbound fluorescent proteins. Recently, RNA-based fluorogenic aptamers have been used for advanced live-cell RNA imaging [19‒28]. These fluorogenic RNA aptamers, which evolved in vitro through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, can specifically bind to their cognate fluorogenic dyes and significantly activate their fluorescence [29‒32].

The Malachite Green aptamer was the first fluorescent RNA aptamer, originally developed in 2003 [33,34]. In 2011, the Jaffrey group synthesized a series of fluorogenic HBI analogues and selected an RNA mimic of GFP called Spinach, marking a critical breakthrough in the development of fluorogenic RNA aptamers [35]. Since then, various approaches have been employed to fine-tune and improve the properties of Spinach and its cognate fluorophore molecules, leading to the identification of related aptamers such as Broccoli [36], Corn [37], Beetroot [38], Chili [39] and Squash [40] aptamers. In addition, multiple other fluorescent RNA aptamer systems have been isolated and characterized, including cyanine dye-based aptamers [41‒44], contact quenching-based aptamers [45‒52], and spirolactonization-based aptamers [53,54]. Recently, two novel fluorogenic aptamers, Pepper and Clivia, which feature high cellular brightness, good photostability and multiple spectral properties, have been developed, greatly enriching the available toolbox for RNA imaging [55‒57].