In the rapidly evolving field of RNA biology, the ability to visualize RNA molecules inside living cells with exquisite sensitivity and specificity has become a sought-after goal for scientists worldwide. Over the past decade, RNA-based fluorogenic aptamers have emerged as revolutionary tools, turning previously weak or dark small-molecule fluorophores into bright, fluorescent beacons upon binding. Among these, the Mango aptamer family has garnered widespread attention due to its high affinity for tailored dyes and capability to illuminate RNA molecules with remarkable clarity. Now, a novel advancement in this domain promises to significantly enhance our capacity to image RNA dynamics inside cells through the development of an ultrabright fluorophore — an optimized molecular partner for the Mango II aptamer, discovered through an innovative, structure-guided approach.
Conventional fluorescence technologies for RNA visualization rely heavily on fusion proteins or fluorescent tags that can introduce artifacts or perturb native RNA function. In contrast, fluorogenic aptamers like Mango II, which are short RNA sequences that bind nonfluorescent dye molecules and activate their fluorescence, offer unparalleled specificity and minimal interference. Despite these advantages, the crux of progress in the field hinges on discovering brighter, more selective, and tighter-binding ligands that can function efficiently inside the complex milieu of living cells. Addressing these challenges, a team led by Yang, Prestwood, and Passalacqua has unveiled SALAD1 — a cutting-edge fluorophore intelligently designed to surpass existing Mango II dye complexes by many folds in brightness and binding affinity.
The journey to SALAD1’s discovery is itself a testament to the power of rational molecular design combined with high-throughput screening. The investigators employed a structure-informed, fragment-based small-molecule microarray platform, a technique where thousands of chemically distinct fragments are systematically screened for binding efficiency and fluorescent activation potential. Using the detailed crystallographic data of the Mango II aptamer binding pocket, they refined candidate molecules iteratively to optimize interactions without compromising the critical π-conjugated system of the fluorophore responsible for light absorption and emission. This multifaceted approach led to the identification of SALAD1, a dye that binds Mango II with subnanomolar affinity, an unprecedented feat that ensures robust, stable complex formation even at minute cellular concentrations.
SALAD1’s photophysical properties mark a substantial leap forward. When bound to Mango II, it exhibits fluorescence intensities approximately 3.5 times greater than the well-characterized Mango II-TO1–biotin fluorophore system, a current benchmark in RNA imaging. This remarkable brightness facilitates the detection of low-abundance RNA species, enabling researchers to track subtle changes in RNA localization and dynamics in real time. Moreover, SALAD1’s enhanced affinity prevents premature dissociation— a common limitation of fluorogenic dyes— thereby improving signal-to-noise ratios and reducing false-positive visualization events in cellular contexts.
Insight into the molecular underpinnings of SALAD1’s superior performance was gained through high-resolution X-ray crystallography. Structural snapshots revealed an exquisitely fitting molecular architecture where SALAD1 fully occupies the Mango II binding pocket with enhanced shape complementarity and electrostatic interactions. Notably, SALAD1 establishes a unique bonding interaction involving a potassium ion within the RNA-ligand complex, a feature absent in previously characterized Mango fluorophores. This interaction likely stabilizes the complex, contributing to both improved binding affinity and photostability. Understanding these subtle but critical contact points paves the way for future rational modifications aiming at even higher performance fluorogens.
The researchers also fine-tuned RNA-dye molecular recognition elements without disturbing the extended π-electron system central to SALAD1’s fluorescence. Such a delicate balance is essential; alterations to the fluorophore’s electronic structure can drastically affect emission efficiency and spectral properties. By preserving this core while enhancing pocket occupancy and interaction specificity, SALAD1 embodies a design philosophy that leverages both structural precision and photophysical optimization.
Importantly, SALAD1 exhibits excellent cell permeability, an often overlooked yet vital characteristic for live-cell RNA imaging applications. Many promising dyes fail to traverse cellular membranes efficiently, limiting their utility. SALAD1’s membrane crossing capacity, coupled with its high fluorescence upon Mango II binding, ensures it can be introduced into living cells without invasive delivery methods, significantly simplifying experimental workflow. This characteristic further amplifies the potential impact of SALAD1 for dynamic studies of RNA processing, transport, and localization within native cellular environments.
In confocal microscopy experiments, SALAD1 paired with Mango II outperformed its predecessors, delivering crisp, high-contrast images of target RNAs inside living cells. The enhanced brightness and affinity translated into more detectable RNA foci with lower background fluorescence, facilitating the visualization of transient RNA structures and low-expression transcripts that were previously difficult to capture. This capability paves the way for novel investigations into gene regulation, RNA metabolism, and the spatiotemporal orchestration of cellular processes governed by RNA.
Beyond technical performance metrics, the introduction of SALAD1 represents a broader methodological breakthrough—the effective application of fragment-based ligand discovery tailored to RNA targets. While fragment-based approaches have revolutionized protein ligand design, their adoption for RNA has been limited by the intrinsic challenges of RNA structural complexity and dynamics. By successfully adapting this strategy within an RNA aptamer framework, the authors demonstrate a powerful pipeline for next-generation RNA-targeted chemistry, enabling the de novo design of ligands that were previously unattainable.
The implications of this study extend well beyond just the Mango II aptamer system. The principles underpinning SALAD1’s development—exploiting high-resolution RNA structures to guide fragment selection, optimizing molecular interactions for affinity and specificity, and maintaining fluorophore integrity—can be generalized to engineer fluorogenic ligands for a plethora of other RNA motifs and functional classes. Such expansion could revolutionize how scientists interrogate RNA biology across diverse organisms and experimental conditions.
Moreover, the combination of structure-informed design with innovative microarray screening platforms heralds a new era for RNA chemical biology, where the rational synthesis of bespoke RNA-binding molecules becomes routine. This facilitates the targeting of complex RNA architectures involved in disease, enabling both diagnostic and therapeutic innovations. The discovery of SALAD1 thus represents a foundational step toward constructing a versatile toolkit of RNA-activated fluorophores and functional probes with broad biomedical and research relevance.
The work by Yang and colleagues emphasizes the power of interdisciplinary collaboration, merging expertise in structural biology, chemistry, molecular biology, and imaging technologies to solve an enduring challenge in RNA research. Their approach underscores the synergy between computational modeling, empirical screening, and atomic-level structural validation, illustrating how modern science is increasingly convergent and integrative.
Looking forward, further exploration of SALAD1 derivatives with altered spectral properties or environmental sensitivities could unlock even more sophisticated RNA imaging modalities, including multiplexed and super-resolution approaches. Additionally, engineering genetically encoded RNA aptamers with expanded binding repertoire and enhanced structural stability holds promise to expand the synthetic biology applications of ultrabright RNA fluorophores like SALAD1.
In summary, the structure-informed discovery of the ultrabright SALAD1 fluorophore for the Mango II RNA aptamer system marks a landmark achievement in the field of RNA imaging. By achieving a delicate equilibrium of molecular recognition, fluorescence enhancement, and cellular compatibility, this study sets a new gold standard for fluorogenic RNA aptamer/dye pairs. Such breakthroughs not only deepen our understanding of RNA biology but also equip researchers with potent tools to visualize and manipulate RNA with extraordinary precision, heralding impactful advances in molecular diagnostics, gene regulation studies, and therapeutic development.
Subject of Research: RNA-based fluorogenic aptamers and fluorophore design for enhanced intracellular RNA imaging.
Article Title: Structure-informed design of an ultrabright RNA-activated fluorophore.
Article References:
Yang, M., Prestwood, P.R., Passalacqua, L.F.M. et al. Structure-informed design of an ultrabright RNA-activated fluorophore.
Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01832-w
Image Credits: AI Generated
