Lighting Up Endogenous RNA: Fluorescent Aptamer Sensors for Live-Cell Imaging

Abstract

Endogenous RNAs orchestrate cellular processes through precise spatiotemporal regulation, and live-cell imaging of these molecules is essential for dissecting their roles in gene expression, disease progression, and cellular homeostasis. While traditional methods rely on exogenous labeling or fixation, fluorescent RNA aptamer-based sensors have emerged as transformative tools for visualizing endogenous RNAs in their native context. These sensors allow real-time tracking of mRNA localization, miRNA activity, and RNA–protein interactions with minimal disturbance. However, challenges such as the requirement for exogenous fluorogenic dyes, limited brightness, photostability, and target specificity in complex cellular environments hinder their wider application. This review provides a comprehensive overview of recent advances in fluorescent RNA aptamer-based sensors, discussing the design principles, mechanisms of fluorescence activation, and their application in live-cell RNA imaging. We also address the current limitations and future directions for improving these sensors, highlighting their transformative potential in RNA biology and their implications for diagnostic and therapeutic strategies.

1. Introduction

RNA molecules are critical in cellular and gene expression regulation. They adopt specific structures to impact transcription, RNA processing, translation, and turnover. RNA types include mRNA, tRNA, rRNA, and regulatory RNAs, with mRNA-encoding proteins and other RNAs involved in chromosomal organization, DNA repair, and metabolic regulation. , Some RNAs also have catalytic activity, similar to enzymes. RNA structures play a significant role in various physiological and pathological processes. The dynamic behavior of endogenous RNAs, such as mRNA trafficking to specific subcellular compartments or miRNA-mediated gene silencing, underpins critical biological events ranging from embryogenesis to cancer metastasis. For instance, the asymmetric distribution of β-actin mRNA in migrating fibroblasts regulates cell polarity, while dysregulated miRNA-21 expression in tumors drives oncogenic signaling. Imaging the localization and dynamics of RNAs helps uncover their function and reveals novel mechanisms at single-cell resolution. RNA imaging has highlighted the importance of post-transcriptional mechanisms like mRNA localization, but challenges remain due to technological limitations and RNA biology complexity. While protein imaging is routine, RNA imaging in live cells has been less explored, though recent advances in RNA binding proteins and chemical biology have improved visualization tools. Notwithstanding these advancements, unresolved challenges persist in achieving high-fidelity real-time visualization of endogenous RNA within intact mammalian systems, alongside comprehensive monitoring of spatiotemporal RNA dynamics and functional interactions in native biological contexts.

Current RNA imaging techniques are primarily divided into fixed and live-cell RNA imaging. Unlike FISH-based fixed-cell imaging, live-cell RNA imaging provides temporal information, enabling the analysis of gene expression regulation and dynamic RNA processes. − This approach establishes a multidimensional analytical framework for investigating RNA lifecycle processesspanning biosynthesis, spatial distribution, regulatory mechanisms, intracellular retention, and disposal pathwaysin diverse cellular populations. , In vivo RNA imaging technologies fall into two main categories: fluorescent protein-based systems, such as the MS2/MCP, − PP7/PCP system, − box B/λN22 system, − PUM-HD system, , CRISPR/dCas13 system, , Pepper/tDeg system, fluorescent dye-based technologies, including molecular beacons − and fluorescent RNA aptamer/fluorescent dye systems. Each live-cell RNA imaging technology offers distinct advantages and limitations, shaping their suitability for different biological questions. MS2/PP7-like systems rely on coexpression of an RNA-binding protein fused to a fluorescent protein (FP) and require insertion of multiple cognate RNA stem-loops into the target RNA. They provide high specificity and brightness from the FP but necessitate significant genetic modification of both the RNA and the cell, potentially perturbing RNA function, and suffer from background from unbound FP. Molecular Beacons (MBs) are dual-labeled oligonucleotide probes (fluorophore-quencher) that fluoresce upon hybridization to a complementary target RNA. They require no genetic modification and can be delivered synthetically. While offering rapid detection, they suffer from poor cellular delivery, nuclease sensitivity, potential off-target binding, and inability to distinguish nuclear/cytoplasmic RNA effectively. CRISPR/dCas13-FP systems use a catalytically dead Cas13 protein fused to an FP and programmed with a guide RNA (gRNA) to bind specific RNA sequences. They offer high programmability and specificity without modifying the target RNA itself but require delivery/expression of large dCas13-FP and gRNA components, and FP background can be significant. Fluorogenic Dyes with RNA-Binding Proteins combine a genetically encoded protein component that degrades the fluorophore unless bound to its target RNA sequence. They offer high contrast but involve complex protein engineering and delivery. However, the development of fluorescent RNA aptamers has been a significant breakthrough in recent years, offering noninvasive insights into RNA biology.

When tracing the origins of the fluorescent aptamer/dye system, green fluorescent protein (GFP) is an essential mention. In live-cell protein imaging, the success of GFP established the paradigm of genetically encoded, target-specific fluorescence activation. This motivated the search for analogous tools for RNA. A pivotal conceptual leap was the proposal and realization of creating ’RNA mimics of GFP’. Using SELEX against the GFP fluorophore derivative 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), Jaffrey and his team isolated the first practical fluorescent RNA aptamer. They named it ″Spinach″ due to its green fluorescence. Spinach demonstrated that RNA scaffolds could specifically bind to and dramatically enhance the fluorescence of small fluorophores that would otherwise be dim, thereby enabling genetically encoded RNA tagging. However, initial Spinach variants encountered issues such as misfolding and magnesium dependence. This spurred intensive engineering efforts, resulting in improved derivatives such as Baby Spinach, iSpinach and the more robust Broccoli. Parallel exploration identified aptamers that bind to distinct fluorophores, expanding the color palette: The red-shifted Mango aptamers that bind thiazole orange (TO) derivatives, the orange/red SRB-2/SR-DN system, , the yellow Corn aptamer that binds DFHO, the near-infrared SiRA, the cyan Squash, the dimeric Beetroot, and, most recently, the exceptionally bright Pepper aptamers that span green to red, Okra, and Clivia. This continuous evolution, driven by the need for brighter, more stable and more spectrally diverse systems that are easier to deliver, has firmly established fluorescent RNA aptamers as indispensable tools for live-cell RNA biology.

RNA aptamers offer advantages over antibodies, including ease of synthesis, chemical stability, low molecular weight, specific targets with high affinity and specificity and lack of immunogenicity. These aptamer-based fluorescent sensors provide exciting opportunities for sensitive detection and quantification of various analytes, including cancer biomarkers, small molecules, and other biomolecules. However, exogenous RNA labeling requires complex genetic manipulation, which may affect RNA activity. As research progresses, improvements are being made to enable the imaging of endogenous RNA in its natural state without genetic manipulation. , Recent breakthroughs in aptamer engineeringsuch as split-probe designs and fluorogenic dye optimizationhave further enabled the visualization of low-abundance miRNAs and single-molecule mRNA movements, positioning these sensors as indispensable tools for unraveling RNA-mediated mechanisms in live cells. It would be helpful to visualize RNAs in cells − and enable the visualization of RNA in situ, which is key to studying the temporal and spatial regulation of gene expression during development. Crucially, while these sensors require the addition of exogenous fluorogenic dyes, they enable visualization of endogenous, unmodified RNA transcripts in their native cellular context, circumventing the need for genetic manipulation of the target RNA itself. This review summarizes recent advances in fluorescent RNA aptamer-based sensors for imaging endogenous RNA in live cells, highlighting their applications, challenges, and potential in RNA biology and molecular diagnostics.

2. Fluorescent RNA Aptamers: Design and Mechanism

Fluorescent RNA aptamers are single-stranded RNA molecules that bind specific fluorogenic ligands, enhancing fluorescence. These aptamers are valuable tools for RNA detection, imaging, and biosensing applications. The design of fluorescent RNA aptamers typically involves in vitro selection methods such as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to isolate sequences with desired binding and fluorescence properties. The mechanism of fluorescence enhancement in RNA aptamers often relies on base quadruples (tetrads) to constrain the photoexcited chromophore. Structural studies reveal that these quadruplexes form rigid binding pockets. The planar chromophore of the dye stacks between the faces of these tetrads or within a cavity formed by them. This constrained environment significantly restricts rotational and vibrational freedom of the dye molecule in its excited state, a major pathway for nonradiative energy loss. This restriction minimizes internal conversion and promotes radiative decay, leading to the dramatic fluorescence enhancement characteristic of these aptamers. This structural feature is shared among unrelated fluorogenic RNAs, highlighting its importance in fluorescence activation. Additionally, some aptamers can be designed as ″structure-switching signaling aptamers,″ which undergo conformational changes upon ligand binding, resulting in increased fluorescence.

2.1. Principles of RNA Aptamer Design for Fluorescence

RNA aptamers designed for fluorescence applications typically employ structure-switching mechanisms and specific binding interactions to generate detectable signals. These aptamers can be rationally designed or selected through in vitro evolution to bind nonfluorescent dyes and activate their fluorescence, making them valuable tools for biosensing and molecular imaging (Figure ). The design principles of fluorescent RNA aptamers often involve the use of base quadruples (tetrads) to constrain the photoexcited chromophore, as revealed by 3D structural studies of fluorogenic RNAs crucial for efficient fluorescence activation.

1.

Fluorescent RNA aptamer toolkit.

For fluorescence-based applications, RNA aptamers can be designed to interact with a fluorophore or a fluorescent dye that enables them to emit light upon excitation. The aptamers are usually modified by attaching a fluorophore at specific positions within the RNA sequence, often through covalent linkage at the RNA’s 3′ or 5′ ends. Alternatively, RNA aptamers can be designed to include specific fluorescent probe structures, such as the 2-aminopurine (2-AP) nucleotide, which fluoresces upon binding to a specific target molecule. The optimization of RNA aptamers for fluorescence typically involves the balancing of factors such as binding affinity, selectivity, and fluorescence intensity to achieve optimal performance in real-time imaging. Understanding the precise structural basis of dye binding and fluorescence activation, as revealed by crystallography and NMR studies, is crucial for rational engineering efforts aimed at improving these parameters.

2.2. Mechanism of Fluorescence Activation upon RNA Binding

Fluorescence activation upon RNA binding involves complex mechanisms that vary depending on the specific RNA-fluorophore system. The fluorescence mechanism of RNA aptamers largely depends on incorporating fluorophores that either emit fluorescence directly upon excitation or undergo conformational changes that activate fluorescence. Different RNA aptamers employ distinct mechanisms for fluorescence activation (Table ).

• Fluorophore-quencher pairs: Some RNA aptamers are designed to bring a fluorophore and a quencher nearby when bound to their target, leading to fluorescence quenching in the free state. Upon target binding, the aptamer undergoes a conformational change that separates the fluorophore from the quencher, restoring fluorescence. This mechanism is commonly seen in molecular beacons, where the RNA aptamer functions as a “sensor”.

• Fluorophore stabilization: In other cases, the fluorophore is embedded within the aptamer structure so that its fluorescence is initially suppressed due to interactions with the RNA itself or neighboring bases. Binding to a target induces a structural rearrangement that releases the fluorophore from its quenching environment, leading to a fluorescence ″turn-on″ effect. This design is helpful in bioimaging for detecting specific cellular biomarkers or monitoring molecular interactions in live cells.

• Aptamer-target-induced conformational changes: RNA aptamers may undergo large-scale conformational changes upon binding to a specific target. These structural transitions can lead to the exposure or de-exposure of fluorophores embedded in the aptamer, causing fluorescence to either increase or decrease, depending on the nature of the binding molecules. This mechanism is central to the functionality of RNA-based sensors in imaging applications.

1. Key Photophysical Properties and Structural Features of Some Major Fluorescent RNA Aptamer–Dye Pairs.

Aptamer	Fluorophore	Ex (nm)	Em (nm)	Quantum Yield (Φ)	Brightness (ε × Φ)	K d (nM)	Structure
Spinach	DFHBI	469	501	0.72	∼9,500	540	G-Quadruplex
Broccoli	DFHBI-1T	469	501	0.94	∼12,000	360	G-Quadruplex
Mango-IV	TO1-Biotin	510	535	0.97	∼45,000	3.2	G-Quadruplex
Corn	DFHO	505	545	0.61	∼18,000	380	Dimeric
Pepper	HBC530	485	530	0.85	∼28,000	4.7	Tuning-fork
SRB-2	SR-DN	579	596	0.45	∼15,000	120	Variable
Beetroot	DFHO	515	560	0.49	∼14,000	110	Dimeric
Okra	Okra-DF	510	560	0.99	∼58,000	1.8	Not reported
Clivia	Clivia-650	645	670	0.42	∼16,000	25	Not reported

The common thread among these mechanisms, underpinned by structural insights, is the aptamer’s ability to impose a specific, rigid conformation on the fluorophore upon binding. This conformational constraint, often facilitated by interactions within pockets formed by base quadruples or other tertiary structures, effectively suppresses nonradiative decay pathways that dissipate energy as heat. Consequently, the probability of radiative decay increases dramatically, enabling sensitive detection.

3. Fluorescent RNA Aptamer-Based Sensors for Imaging Endogenous RNA

Imaging endogenous RNA within live cells has become an essential tool for understanding the dynamics of RNA biology in real time. The ability to visualize RNA molecules directly offers insights into critical cellular processes, including transcription, splicing, transport, localization, and degradation. Fluorescent RNA aptamers have enabled the detection and visualization of endogenous RNA species with high spatial and temporal resolution (Table ).

2. RNA Aptamer-Based Sensors Mentioned in the Text That Can Be Used for Imaging Endogenous RNA in Live Cells.

New System	Basic System	Fluorophore	Ex (nm)	Em (nm)	Applications
FASTmiR	Spinach	DFHBI	469	501	miR122, miR171
AiFc	Broccoli	DFHBI	469	501	β-actin, CFL1, GAPDH mRNA
CHARGE	Broccoli	DFHBI-1T	472	507	miR21
INSIGHT	Broccoli	DFHBI-1T	472	507	survivin RNA
RIFC	Corn	DFHO	505	545	miR21
Corn-Based Fluorescent Light-Up Biosensor	Corn	DFHO	505	545	lncRNA MALAT1
iPepper	Pepper	HBC530	485	530	β-actin, CTTN, GAPDH, TUBB3, EHMT2, CLASP1, and ARHGEF11 mRNA
Light-Up RNA Sensor	SRB2	SR-DN	579	596	miR21
SR Aptamer RNA Sensor with GFP	SRB2	SR-DN	579	596	survivin RNA
Dual-Color Light-Up RNA Sensor	SRB2 and Mango	SR-DN and TO1-biotin	579	596	miR224
			510	535

3.1. Imaging Endogenous mRNAs

The ability to track endogenous mRNAs in live cells is critical for understanding their roles in cellular polarization, stress responses, and disease. Traditional methods such as MS2-GFP require genetic modification, which may alter RNA dynamics. Fluorescent RNA aptamers overcome this limitation through direct, label-free imaging.

3.1.1. Aptamer-Initiated Fluorescence Complementation Method (AiFc)

Green fluorescent protein (GFP) emits green fluorescence, and primarily used in advance bioscience research as in vivo reporter protein. GFP contains a 4-hydroxybenzimidazolinone (HBI) fluorophore. HBI and denatured GFP obtained by chemical synthesis are nonfluorescent and only upon refolding the folded GFP protein specifically contact the fluorophore, thereby preventing intramolecular motion and restoring GFP fluorescence. 2011, Jaffrey et al. designed a new HBI derivative 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) was screened for its corresponding RNA aptamer by exponential enrichment ligand phylogenetic techniques (SELEX) and named ″Spinach″ because of its green color of luminescence. Spinach was shown to fuse with the 3′ end of 5S, a noncoding RNA, and effectively label it, resulting in the observation of green fluorescence in cells shortly after Spinach transcription. Following the discovery of Spinach and DFHBI, the system was continuously refined. Additional DFHBI analogs with distinct fluorescence spectra were developed, and various Spinach aptamer variants. Broccoli, a 49nt variant of Spinach, emerged as an improved version due to its reduced dependence on magnesium ions, higher intracellular folding efficiency, and increased brightness, which has gained significant attention (Figure ).

2.

Fluorescent RNA aptamer-based on broccoli for imaging endogenous RNA. Adapted with permission from ref . Copyright 2016 Elsevier Ltd.

To use fluorescent aptamers to image endogenous mRNAs, researchers have explored a variety of applications. The strategy of disassembling the intact RNA aptamer to make the fluorescence disappear and then designing it to bind to the target RNA close to each other to restore the aptamer structure and thus direct the fluorescence to turn on is very effective. The ″Split-Spinach″ concept was proposed by Rogers et al. in 2015, who found that Spinach aptamers could be split in half at a suitable site in a third stem, and fluorescence could be restored after assembly. However, although he showed that splitting Spinach aptamers is a potential tool for monitoring artificial or natural RNA, he did not apply it. 2018, Wang et al. reported an aptamer-initiated fluorescence complementation method (AiFc) for RNA imaging. They split an intact RNA aptamer into two parts at suitable sites, each part tandem with a part of the complementary sequence of the target RNA, and only in the presence and hybridization of the target RNA can the two luminescent RNA aptamer parts be drawn together thereby restoring them to their fluorescent form. They split Baby Spinach and Broccoli by screening cleavage sites and successfully used them for imaging several different endogenous mRNAs. The AiFc splitting strategy essentially reduces the presence of background signal, and the hybridization region of the target RNA can be replaced with a variety of RNAs, significantly increasing its versatility (Figure ). To use AiFc to other fluorescent RNA aptamers should potentially expand its application in RNA multicolor imaging.

3.

Fluorescent RNA aptamer-based on broccoli for imaging endogenous RNA. (A) Design of broccoli-based AiFC probes. (B) AiFC probe imaging of mRNAs. Adapted with permission from ref . Copyright 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

3.1.2. Inert Pepper System (iPepper)

Pepper-based fluorescent RNA aptamer sensors have emerged as powerful tools for imaging and detecting various targets in live cells. These sensors exhibit superior brightness, photostability, and signal-to-background ratio compared to other fluorescent light-up RNA aptamers (FLAPs). The Pepper aptamer folds into a unique monomeric non-G-quadruplex tuning-fork-like architecture, which allows for efficient fluorophore binding and activation. Chen et al. developed the fluorescent dye HBC, featuring a rigid electron acceptor and a strong electron donor. HBC remains nonfluorescent in solution but regains fluorescence upon binding to the Pepper RNA aptamer. By modifying donor–acceptor properties, they created HBC analogs emitting fluorescence from green to red, all compatible with Pepper. Their experiments demonstrated Pepper’s potential for real-time RNA tagging and imaging, offering improved stability, higher binding affinity, and enhanced fluorescence compared to previous aptamers.

Wang et al. introduced the inert Pepper system (iPepper), modifying the original aptamer by adding complementary sequences at its 5′ and 3′ ends (Figure ). This design enables fluorescence activation only in the presence of target RNA. They also developed iPepper tandem arrays, significantly enhancing fluorescence intensity and enabling effective imaging of low-expression endogenous mRNAs.

4.

Fluorescent RNA aptamer-based on pepper. (A) Schematic illustration of inert pepper (iPepper) aptamer. (B) Fixed HeLa cells with and without transfection were treated with the Cy3 probe targeting β-actin and then imaged in the presence of HBC. Adapted with permission from ref . Available under a Creative Commons CC BY license. Copyright Wang, Q.; Xiao, F.; Su, H.; Liu, H.; Xu, J.; Tang, H.; Qin, S.; Fang, Z.; Lu, Z.; Wu, J.; et al. 2022.

3.1.3. INSIGHT System

To amplify the signal while preserving the localization information of the target mRNAs, Ren et al. designed a genetically encoded in situ amplification strategy called INSIGHT. By dividing the broccoli into two parts, using a hybridization chain reaction (HCR) related design, so that the system trigger cascade hybridization of the hairpin structure only in the presence of the target RNA binding, thus activating the amplified fluorescent signal, and at the same time the designed broccoli strand will be associated with the target RNA strand to achieve real-time localization of the target RNA. The INSIGHT system has been demonstrated to apply to RNA imaging of bacterial cells and living mammalian cells (Figure ).

5.

Fluorescent RNA aptamer-based on broccoli. (A) Design of INSIGHT system. (B) Imaging the subcellular location of mRNAs in cells. Adapted from ref . Copyright 2020 American Chemical Society.

3.1.4. Co-expressing RNA Sensor Based on SRB-2

The SRB-2 aptamer, initially identified against sulforhodamine B, exhibits exceptional adaptability in attaching to a range of dyes displaying diverse hues, leading to either an enhancement or suppression of fluorescence. This characteristic positions SRB-2 as a promising candidate for fluorescence microscopy and biological analyses. By conducting a thorough examination of fluorophore structural components and dissociation constants, researchers have successfully engineered a novel, vivid, orange fluorescent turn-on probe termed TMR-DN. This probe facilitates nonwash live-cell RNA imaging, featuring a notably enhanced signal-to-background ratio. In a study by Sunbul et al., a sensor was created in which specific RNA aptamers engage with fluorophore-quencher conjugates to regenerate fluorescence. The researchers employed sulfonyl rhodamine B (SR) and its corresponding aptamer, SRB-2, in conjunction with dinitroaniline (DN) as a triggering agent, thereby establishing the SRB-2/SR-DN system.

Ying et al. designed an RNA sensor based on a single promoter system for mRNA ratio imaging. By coexpressing SRB-2 aptamer and GFP in a single transcript, they avoided the issues of signal misalignment between dual promoters (Figure ). To ensure proper fluorescence, they incorporated a substrate site cleaved by Dicer enzyme and an mRNA-stabilizing triplex to prevent rapid GFP mRNA degradation. This system improved proportional imaging and provided a more reliable approach for RNA aptamer-GFP coexpression.

6.

Fluorescent RNA aptamer-based on SRB2 for imaging endogenous RNA. (A) Illustration of plasmid design and the RNA sensor from a splittable fusion transcript with GFP for ratio metric mRNA imaging. (B) Fluorescence images of mRNA in cells. Adapted with permission from ref . Copyright 2020 Wiley-VCH GmbH.

3.2. Detecting miRNAs in Live Cells

MicroRNAs (miRNAs) regulate gene expression at post-transcriptional levels, and their dysregulation is linked to diseases such as cancer. Fluorescent aptamers offer a powerful tool for real-time miRNA detection.

3.2.1. Sensor “Pandan”

To enable the imaging of natural RNA without genetic manipulation, researchers have explored modifications to fluorescent RNA aptamers. Aw et al. introduced additional stem-loops into Spinach and changed its 3′ and 5′ ends to include two sequence-variable stem-loops encoding complementary sequences of the target microRNA. Only after binding target microRNA, the reconstruction of the RNA scaffold can be reconstituted for DFHBI binding and fluorescence emission, which finally triggers a substantial enhancement of fluorescence. They named this new circular bimolecular sensor ″Pandan″ and accomplished the detection of miRNA in vitro. Although Pandan did not complete the detection of intracellular miRNAs, this hybridization-induced structural repair of RNA aptamers and, thus, restoration of fluorescence provided a new idea for aptamers uses.

3.2.2. FASTmiR Probes

Huang et al., designed FASTmiR by using similar strategy of hybridization-induced structural repair as Pandan. They accomplished rapid quantification of microRNAs in vitro and direct detection and localization of microRNAs in vivo by generating a tandem repeat sequence of a sensor stabilized by a three-way junction (SWJ) based on Spinach. The system has been validated to complete detection using both miR122 and miR171 as targets, demonstrating its potential to detect various microRNAs with modified binding site region sequences specifically.

3.2.3. Sensor Based on the SRB-2/SR-DN System

Based on the SRB-2/SR-DN system, Ying et al. reported a microRNA sensor for live mammalian cell imaging, incorporating a complementary sequence of the target microRNA into the SRB-2 aptamer. This disrupted the aptamer’s structure, preventing SR-DN fluorescence recovery until the target microRNA was present. The aptamer was protected with a tRNA structure to ensure stability in cells. This system enabled the correct folding of SRB-2 and fluorescence restoration only in the presence of the target microRNA. They also designed a coexpression system with RNA sensors and GFP for proportional imaging of microRNAs in tumor cells.

3.3. Visualization of Other Noncoding RNAs

3.3.1. CHARGE Circuit

Karunanayake et al. designed genetically encoded RNA-based catalytic hairpin assembly circuit, CHARGE, for sensitively detecting low-abundance RNA in cells. They divided Broccoli into two nonfluorescent parts, which were bound to the ends of two catalytic hairpin assemblies (CHA), and the target RNA served as a catalyst. The presence of the target RNA causes the two catalytic hairpins to assemble and simultaneously restore the fluorescence of Broccoli (Figure ). In contrast, the target RNA catalyze the activation of tens to hundreds of Broccoli fluorescence, enabling highly sensitive detection of sRNA, and the circuit has been demonstrated in bacterial cells. However, although the CHARGE circuit is highly sensitive, but cannot provide information about the location of the target RNA because the target RNA is shed after the end of catalysis.

7.

Fluorescent RNA aptamer-based on broccoli for imaging endogenous RNA. (A) Schematic of the CHARGE circuit. (B) Confocal fluorescence imaging of cells expressing H1+H2+MBSgrs. Adapted from ref . Copyright 2018 American Chemical Society.

3.3.2. Corn-Based Fluorescent Light-Up Biosensor

Song et al. synthesized DFHO, an RFP fluorophore mimic, based on RFP’s structure. , DFHO, containing a N-acyl imine substituent, demonstrated lower background fluorescence, low cytotoxicity, and enhanced photostability, making it ideal for cellular applications. A fluorescent RNA aptamer, named ″maize″ due to its yellow fluorescence, was developed through SELEX and truncation analysis. A distinctive feature of the Corn aptamer is its dimeric structure, which is crucial for DFHO fluorescence activation. Based on this, Gu et al. designed the RIFC probe, which splits Corn into two parts, each containing the target RNA’s recognition sequence. When the target RNA is present, the aptamers self-assemble and activate DFHO fluorescence. Verified for imaging microRNAs in living cells, the RIFC probe is versatile and can be adapted to image various RNAs by replacing the target sequence, making it a ″plug-and-play″ tool. Corn-based fluorogenic RNA aptamer sensors offer significant advantages in cellular imaging and biosensor applications. The ability to convert Corn into a small-molecule-regulated fluorescent biosensor, as demonstrated with the S-adenosylmethionine (SAM) sensor, and its use in split aptamer systems showcase the versatility of this aptamer.

Qian Zhang et al. designed a biosensor using a cyclic cleavage reaction to amplify RNA aptamer-Corn for the sensitive detection of lncRNA. By employing magnetic beads (MBs) for target enrichment and separation, they achieved highly sensitive quantification of lncRNA at femtomolar levels, with the ability to distinguish tumor cells from normal ones. The approach could be easily adapted to detect various lncRNAs by altering the capture probe sequences, offering a new tool for molecular diagnostics. These developments provide new opportunities for real-time visualization and mechanistic analysis of nucleic acids in living cells, with potential applications in tumor-associated RNA detection and RNA/RNA interaction monitoring (Figure ).

8.

Fluorescent RNA aptamer-based on corn for imaging endogenous RNA. (A) Design of corn-based fluorescent light-up biosensor. (B) DFHO fluorescence signals induced by cells. Adapted from ref . Copyright 2023 American Chemical Society.

3.4. Multiplexed RNA Imaging with Dual-Color Aptamers

Dual-color fluorescent RNA aptamer sensors offer significant advantages for RNA detection and imaging, including ratiometric analysis, improved sensitivity, and the potential for multicolor imaging. These systems have shown promise in various applications, from real-time studies in cell and synthetic biology to the detection of specific RNA targets in living cells.

3.4.1. FRET-Enabled Dual-Color System

Early generation RNA quantification systems relied on coexpressed GFP mRNA under unified transcriptional control as calibration modules. Nevertheless, the photophysical constraints and spatial resolution limitations inherent to GFP-based references prompted the development of next-generation RNA imaging platforms. Contemporary solutions employ dual-output RNA aptamer systems that implement FRET-enabled ratiometric detection, significantly improving sensitivity through coordinated fluorophore pairing.

Pioneering work by Zhang et al. engineered a conformation-switching aptamer-fluorophore assembly exhibiting cyan-to-NIR spectral conversion upon target binding, permitting quantitative NIR-based RNA surveillance with single-cell resolution across heterogeneous expression gradients. The progression toward multimodal detection has enabled strategic construction of chimeric aptamer assemblies. By implementing Watson–Crick mediated fusion of Mango-IV and iSpinach aptamers, investigators achieved cross-aptamer FRET activation, laying the groundwork for multiplexed RNA tracking systems that resolve colocalized transcripts with spatial precision.

3.4.2. Dicer-Mediated Proportional Imaging

Dou et al. developed a ratiometric biosensing platform leveraging complementary fluorescent aptamers for microRNA quantification in mammalian systems. Their design incorporated Mango aptamer as a stable calibration unit paired with target-responsive SRB-2 aptamer, interconnected via a Dicer-sensitive dsRNA bridge enabling endogenous enzyme-mediated signal modulation. The sensor restores fluorescence in both Mango and SRB-2 upon target microRNA binding and Dicer cleavage, enabling proportional imaging (Figure ). This strategy minimizes interference and provides a new approach for RNA imaging in living cells, offering potential for building more RNA imaging platforms.

9.

Genetically encoded dual-color light-up RNA. (A) Scheme for the genetically encoded sensor. (B) Images for cells transfected with the sensor. Adapted from ref . Copyright 2021 American Chemical Society.

3.5. Super-Resolution Imaging with Fluorescent RNA Aptamers

Super-resolution (SR) microscopy techniques break the diffraction limit of light, enabling visualization of cellular structures and biomolecule dynamics at the nanoscale. Applying these techniques to RNA imaging holds immense promise for uncovering the true spatial organization and dynamics of transcripts, such as within stress granules or at transcription sites. However, this imposes stringent requirements on the fluorescent probe: high photon output, excellent photostability, and efficient labeling. While early FRAPs struggled to meet these demands, recent advances have yielded aptamers capable of SR imaging. The SiRA (Silicon Rhodamine-Binding Aptamer) aptamer, binding the cell-permeable dye SiR, demonstrated exceptional photostability and single-molecule brightness, enabling robust dSTORM imaging of mRNA distribution and dynamics in fixed and living cells with ∼ 20 nm resolution. SiRA revealed the nanoscale organization of NEAT1 lncRNA within paraspeckles. The exceptional brightness and photostability reported for Okra and Clivia suggest these newer aptamers are also highly promising candidates for pushing the boundaries of live-cell RNA SR imaging, potentially enabling longer trajectories and higher localization precision for single RNA molecules. Combining SR-capable aptamers with advanced microscopy techniques will be crucial for dissecting the nanoscale architecture and dynamic interactions of the RNA transcriptome.

4. Advantages over Traditional RNA Imaging Methods

Fluorescent RNA aptamer-based sensors offer several advantages compared to traditional RNA imaging methods, such as in situ hybridization and RNA probes.

4.1. Real-Time, Dynamic Imaging of Endogenous RNA

Fluorescent RNA aptamer-based sensors enable real-time, continuous monitoring of endogenous RNA molecules in live cells. Unlike traditional methods that often rely on fixed samples and provide only static one-time observations, RNA aptamers allow dynamic observation of RNA behavior, interactions, and localization within their native cellular context.

4.2. High Specificity and Sensitivity

RNA aptamers can be engineered for high specificity, enabling precise targeting of specific RNA sequences. This reduces nonspecific binding and improves imaging accuracy, particularly for low-abundance or transient RNA species. In comparison, traditional methods often lack the sensitivity to detect such targets.

4.3. No Amplification Required

Unlike traditional RNA probes, which typically require amplification techniques to enhance the signal, RNA aptamers do not need any amplification steps. This simplifies the imaging process, reduces potential experimental errors or false signals, and offers a more direct and reliable method of detecting RNA.

4.4. Enhanced Versatility and Modifiability

RNA aptamers can be easily modified to improve their properties, such as fluorescence intensity, photostability, and stability. This flexibility allows for the customization of aptamers for various applications, from single-molecule imaging to multiplexed RNA detection, making them a versatile tool in RNA biology research.

5. Challenges, Limitations, and Future Directions

While fluorescent RNA aptamers hold immense promise, their application to endogenous RNA imaging faces unique hurdles. To address the challenges associated with RNA aptamer-based sensors, several strategies are being explored.

5.1. Specificity Issues and Technology Integration

Many endogenous RNAs share high sequence similarity or exist as splice variants. Current aptamers often lack single-nucleotide resolution, leading to cross-reactivity. Also, RNA folding states vary with cellular conditions, potentially occluding aptamer-binding sites or generating false signals.

Integrating RNA aptamer-based sensors with emerging technologies could significantly enhance future advancements in these sensors. For example, coupling RNA aptamers with gene-editing techniques could enable targeted imaging of specific RNA species in genetically modified organisms. Additionally, integrating RNA aptamers with single-cell sequencing technologies could provide unprecedented insights into the spatiotemporal dynamics of RNA expression at the single-cell level, offering new ways for understanding cellular heterogeneity and gene regulation.

5.2. Sensitivity Amplification and Signal Enhancement

Transcripts like transcription factor mRNAs or rare noncoding RNAs exist at ultralow levels (<10 copies/cell). Existing aptamer-dye systems struggle to achieve sufficient signal-to-noise ratios without amplification. Background noise also is a very important question. Autofluorescence from cellular components and nonspecific dye-RNA interactions obscure target-specific signals, particularly in densely packed organelles like nucleoli.

Developing RNA aptamers with enhanced optical properties is crucial to further expanding their utility for imaging. This includes designing aptamers that exhibit red-shifted fluorescence, allowing for deeper tissue penetration and reduced autofluorescence interference. Optimizing photostability, brightness, and signal-to-noise ratios will make these sensors more reliable and practical for complex imaging environments.

5.3. Delivery and Stability Optimization

Current transfection methods may alter RNA expression profiles or induce stress responses, skewing imaging results. Moreover, aptamer degradation by ribonucleases limits long-term observation.

A significant challenge remains the optimization of aptamer stability in vivo. RNA aptamers are prone to degradation in biological environments, which limits their long-term use in diagnostic and therapeutic settings. Strategies to enhance the chemical stability of aptamers and protect them from enzymatic degradation are critical. Furthermore, developing efficient and targeted delivery systems will be essential to ensure the effective and precise targeting of biomolecules without causing off-target effects or degradation.

5.4. Multiplexing and Super-Resolution Imaging

Most aptamer-dye pairs emit in the green spectrum, hindering simultaneous imaging of multiple RNAs. Also, current dual-color systems require stringent spectral unmixing and lack modularity for diverse RNA targets.

The rational design of next-generation fluorogenic probes requires molecular engineering of dyes with enhanced sensitivity, superior photostability, and tunable spectral properties to advance RNA-targeting biosensing systems. Crucially, concurrent expansion of aptamer libraries through directed evolution and computational design will optimize specificity and enhance binding affinities for diverse pathophysiological biomarkers. This synergistic optimization of probe-aptamer pairs translates to diagnostic platforms with improved clinical resolution while expanding their multiplexing capacity across complex biological matrices.

5.5. Clinical Translation and Diagnostic Potential

Fluorescent RNA aptamer-based sensors show immense promise for clinical diagnostics, offering precise, real-time monitoring of biomolecular interactions. These sensors could detect diseases early, facilitate personalized treatment strategies, and enable noninvasive diagnostics. However, translating laboratory research to clinical application demands rigorous validation in complex biological systems, particularly in human clinical trials.

6. Conclusion

Fluorescent RNA aptamer-based sensors are revolutionizing the study of endogenous RNAs by enabling real-time, label-free imaging of mRNA dynamics, miRNA activity, and RNA-protein interactions in live cells. These tools have illuminated previously inaccessible phenomena, such as the phase separation of stress granule-associated mRNAs or the spatiotemporal regulation of oncogenic miRNAs in tumor microenvironments. While challenges in sensitivity, multiplexing, and in vivo compatibility persist, emerging strategiesfrom CRISPR-coupled targeting to NIR-optimized fluorophorespromise to bridge these gaps. As these technologies mature, they will not only deepen our understanding of RNA biology but also pave the way for clinical innovations, such as intraoperative RNA-guided surgery or personalized therapeutics based on real-time RNA profiling. The next decade will undoubtedly witness fluorescent aptamers transitioning from bench-side curiosities to cornerstone tools in both basic research and translational medicine.

All of the data and materials are included in this manuscript.

The authors declare no competing financial interest.