Simulating green fluorescent proteins: The potential of fluorescent aptamers and peptides for biosensing and imaging

2.1. History of GFP

GFP is a 238-amino-acid polypeptide originally discovered in the jellyfish Aequorea victoria by Osamu Shimomura in 1962 [21]. Its intrinsic fluorescence, which does not require enzymatic cofactors or added substrates, made it an attractive model for optical bioimaging. The molecular basis of its function was elucidated in 1992 when Prasher isolated the gene sequence [22], and its independent fluorescence in foreign hosts was validated by Chalfie in 1994 [1]. These foundational studies paved the way for the rational development of alternative fluorescent platforms that replicate GFP-like functionality through various molecular architectures. Among these, RNA and DNA-based aptamers have been designed to mimic the chromophore sequestration observed in GFP. By adopting well-defined 3D folds, these aptamers selectively bind small fluorogenic molecules, activating their fluorescence without the need for translation or post-translational modification [8,23]. Concurrently, peptide-based systems have emerged, leveraging conformational transitions, especially via loop or ring motifs, to regulate the photophysical behavior of embedded dyes [24]. These systems exhibit rapid response kinetics, minimal size, and enhanced compatibility with in vivo imaging conditions.

Another approach involves de novo engineered proteins that can support the folding and stabilization of fluorophore analogs within artificial scaffolds [25,26]. These constructs, designed via computational methods rather than evolutionary selection, offer modularity and tunable photochemical properties, effectively replicating GFP’s encapsulation function in a synthetic context.

Together, these molecular systems broaden the application of fluorescence, transforming it from static reporters to dynamic and reconfigurable sensors. Their development, rooted in GFP but diversified in the approach, advances goals in cellular visualization, molecular tracking, and environmental responsiveness, particularly in contexts that require compact, genetically independent, or orthogonally tunable fluorescence outputs.

2.2. Structure of GFP

The crystal structure of GFP consists of a tightly packed β-barrel made of 11 antiparallel β-strands. At the center of this barrel, an α-helix contains the chromophore and contributes to the structural integrity of the protein through extensive hydrogen bonding interactions [27,28]. GFP’s fluorescence arises from a mature chromophore, formed through the intramolecular cyclization of a tripeptide composed of serine, tyrosine, and glycine at residues 65–67 [1,29]. This chromophore is deeply embedded within the β-barrel scaffold, effectively shielding it from solvent exposure and preventing fluorescence quenching by molecular oxygen [10,27] (Figures 2a-c).

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Figure 2.

Structure of GFP and the chromophore maturation process. (a) Front view of the β-barrel structure of GFP. (b) Axial view showing the chromophore positioned at the center. (c) Fully formed chromophore with atoms labeled by element: red (O), blue (N), green (C), and yellow (H). (d) Maturation steps: folding, cyclization, oxidation, and dehydration.

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The amino acid trio, serine (Ser), tyrosine (Tyr), and glycine (Gly), undergoes a series of aerobic biochemical processes, including folding, cyclization, oxidation, and dehydration, to form the mature chromophore [30,31]. Although the exact sequence of oxidation and dehydration during chromophore maturation is still debated [10,29], high-resolution mass spectrometry data suggest that oxidation occurs before dehydration when oxygen is present [30]. Proper protein folding is a prerequisite for chromophore maturation [29]. During this folding process, the amide nitrogen of glycine at position 67 nucleophilically attacks the carbonyl carbon of serine at position 65, initiating intramolecular cyclization. This leads to the formation of an imidazolone ring, the structure of which has been confirmed through density functional theory (DFT) calculations [32]. The imidazolone ring then reacts with molecular oxygen, promoting the removal of H₂O₂. A subsequent α-β dehydration reaction at tyrosine position 66 creates a double bond with the imidazolone, resulting in the formation of the mature chromophore, 4-hydroxybenzylidene-imidazolinone (HBI) (Figure 2d). Notably, this entire maturation process occurs without the need for enzymatic activity or cofactors [1], considerably enhancing the versatility of FPs. However, this process is temperature-sensitive, with reaction yields notably decreasing at temperatures above 30°C [33].

2.3. Luminescence mechanism of GFP

The luminescence exhibited by GFP is classified as photoluminescence, specifically cold light emission. This luminescence originates from GFP’s unique chemical structure and the transitions between its internal electronic energy levels. The mature chromophore, synthesized through an autocatalytic cyclization mechanism, consists of a bicyclic structure formed by phenolate and imidazolone rings connected by double bonds [34]. In its resting state, the chromophore adopts a nearly planar cis configuration. When excited by incident light, the electronic energy levels of the GFP molecule shift, resulting in an excited state. This causes the chromophore to transition from a planar to a nearly perpendicular orientation [35]. Hydrogen bonds and hydrophobic interactions within the chromophore limit free rotational movement around the double bond, preventing nonradiative transitions and ensuring that the electrons in the excited state efficiently release their stored energy as fluorescence [36-38].

The GFP chromophore can exist in two distinct protonation states: a neutral form (A*-form) and an anionic form (B-form) [39]. This structural duality leads to two characteristic absorption maxima in the UV–Visible spectrum, at approximately 395 and 480 nm. In the A*-form, excitation triggers a proton transfer event, which produces the anionic excited state (I*-form). The subsequent relaxation of the I*-form back to the ground state results in green fluorescence emission [6,40]. This photoinduced proton transfer mechanism is key to GFP’s unique luminescent properties. Both the efficiency of chromophore maturation and its spatial conformation considerably influence GFP fluorescence intensity and lifetime. For instance, Now GFP—a tryptophan-substituted variant—largely remains in the anionic form under neutral pH at 37°C and exhibits a fluorescence lifetime of 5.1 ns, with a brightness roughly 1.3 times higher than that of enhanced GFP (EGFP) [41].

2.4. Variants of GFP

GFP is widely used as a reporter gene and a marker protein, enabling dynamic monitoring of biological processes. However, its chromophore’s slow maturation and temperature-sensitive folding dynamics can reduce the fluorescence yield, limiting its application in advanced settings. To address these challenges, Roger Tsien applied site-directed mutagenesis to specific amino acids within GFP, creating a series of mutants with enhanced photostability and considerably higher fluorescence intensity [42]. Additionally, FPs found in corals have been shown to exhibit red and far-red spectral properties [43]. These proteins not only maintain the typical exogenous activity of FPs but also exhibit fluorescence characteristics similar to GFP (Table 1).

Table 1. Photophysical profiles of GFP and its spectral variants.

Fluorescent protein	Structure	Chromphore	Mutation site	Excitation peak (nm)	Emission peak (nm)	Molar extinction coefficient	Quantum	Luminance (EGFP %)
DsRed			–	558	583	75000	0.79	176
mOrange			The 66th Gln of DsRed→Thr	548	563	71000	0.69	146
EYFP			The 203th Thr of GFP→Tyr	514	527	83400	0.61	151
EGFP			The 65th Ser of GFP→Thr	488	509	56000	0.6	100
ECFP			The 66th Tyr of GFP→Trp	439	476	32500	0.4	39
EBFP			The 66th Tyr of GFP→His	383	445	29000	0.31	27

The structural and emission characteristics of RFP (Protein Data Bank PDB ID:1G7K), OFP (PDB ID:2H5O), YFP (PDB ID:1YFP), GFP (PDB ID:2QLE), CFP (PDB ID:1CV7), and BFP (PDB ID:1BFP) are shown.

EGFP, a widely used variant, has a serine-for-threonine substitution at residue 65 within its chromophore, a modification that enhances its fluorescence properties [39,44]. Compared to conventional GFP, EGFP exhibits a red shift in its excitation peak to 484 nm and emits bright green fluorescence at 508 nm. Notably, EGFP demonstrates increased brightness and faster chromophore maturation at 37°C compared to GFP [39,45]. Enhanced blue fluorescent protein (EBFP) features a histidine substitution for tyrosine at position 66 within its chromophore, resulting in a shifted excitation maximum at 383 nm and a blue emission peak at approximately 445 nm. The emission spectrum of EBFP considerably overlaps with the excitation spectrum of EGFP, making this pair ideal for applications involving Förster resonance energy transfer (FRET). Despite this overlap, the distinct emission spectra of EBFP and EGFP enable the simultaneous labeling and tracking of different target proteins [42]. Enhanced cyan fluorescent protein (ECFP) features a chromophore in which tyrosine at position 66 is replaced by tryptophan, resulting in an excitation maximum at 439 nm and an emission peak centered at 476 nm. ECFP is widely used in live-cell studies of protein–protein interactions and serves as an efficient donor fluorophore in FRET analyses, especially when paired with enhanced yellow fluorescent protein (EYFP) as the acceptor [46]. Compared to EBFP, ECFP offers greater photostability under prolonged illumination. EYFP, derived from GFP through a threonine-to-tyrosine substitution at residue 203, has excitation and emission maxima at 514 and 527 nm, respectively. Advanced EYFP variants, such as mCitrine and mVenus, offer improved resistance to acidic conditions and exhibit reduced quenching effects in the presence of halide ions [47,48]. Red FPs, like DsRed, efficiently mature their chromophores at 37°C and have excitation and emission peaks at 558 and 583 nm, respectively. Mutants derived from DsRed, including mOrange, Honeydew, and mCherry, show enhanced photostability and increased fluorescence intensity [49-51], advancing research in the red–orange spectrum of FPs.

Mature FPs are highly amenable to genetic modification due to their stability and resistance to physicochemical influences [6]. As a result, these proteins have been successfully integrated into a wide variety of biological systems ranging from animals and plants to fungi and yeast, demonstrating their broad utility in both research and applied fields [52,53]. One of the key advantages of FPs is their ability to be genetically fused with target proteins with minimal disruption to native biological activity [54], allowing for effective tracking, monitoring, and localization of protein expression. Protein transport is tightly regulated by specific peptide signals, and when FPs are fused to peptides that target subcellular compartments, they enable precise visualization of structures such as the Golgi apparatus [55], the mitochondrial matrix [56], and the cell nucleus [57]. Additionally, spectral pairing of FPs in FRET systems facilitates the investigation of protein–protein interactions and the development of biosensors to monitor dynamic changes in intracellular ion concentrations [58,59], amino acids [60], and enzyme activities [61].

The use of FPs for rapid and long-term high-resolution imaging has been limited by their susceptibility to photobleaching, as enhancements in photostability often come at the cost of reduced brightness [62]. However, the monomeric yellow FP mGold, identified through Spotlight screening, has overcome this challenge [63]. mGold offers brightness similar to its parent protein MVenvs while providing a fivefold increase in photostability, making it especially useful for time-lapse imaging in research. StayGold, discovered by Miyawaki et al. in the jellyfish Cladonema uchidae, is currently the most photostable FP known. With brightness comparable to mNeonGreen, StayGold enables dynamic, high spatiotemporal resolution imaging of subcellular structures over extended periods [64]. However, its native dimerization limits its effectiveness as a fluorescent marker. This challenge was addressed by using a tandem dimer construct for unidirectional labeling, followed by disruption of the dimer interface to create a monomeric variant. The resulting monomer retains excellent photostability and brightness, with enhanced dispersibility, making it ideal for long-term continuous imaging [65]. Lin et al. further advanced this technology by incorporating StayGold into virus-like particles, enabling high-speed, real-time three-dimensional tracking of viruses in live cells at localization rates of 1,000 events per second for up to 1 h [66]. The StayGold-derived variant mBaojin demonstrates improved fluorescence intensity, accelerated chromophore maturation, and remarkable chemical robustness, making it suitable for super-resolution neuronal imaging in model organisms, including Caenorhabditis elegans and mice [67].

Near-infrared fluorescent proteins (NIR-FPs), derived from bacterial phytochromes (BphPs) [68,69], incorporate biliverdin IXα (BV), a linear tetrapyrrole, as their chromophore [70]. NIR-FPs offer superior tissue transparency and reduced phototoxicity, enabling deeper tissue imaging without causing cellular damage. However, challenges such as low brightness in live cells and a propensity for dimerization remain. Even monomeric variants like IFR1.4 [71] and IFR2.0 [72] tend to dimerize or aggregate when linked to other proteins, limiting their effectiveness as fluorescent tags [68,73]. To overcome this, protein engineering and directed evolution have been employed to convert dimeric NIR-FPs into monomeric forms, thus enhancing their potential for biological imaging applications [74]. Monomeric NIR-FPs, such as emiRFP670 and emiRFP703, exhibit distinct spectral properties, enabling dual-color imaging in live cells [75]. mIFP663, with a peak excitation wavelength of 633 nm, allows for imaging of subcellular structures and viral proteins without disrupting localization or replication. This overcomes the common challenge of reduced brightness in monomeric NIR-FPs under standard laser excitation [76].

The development of these NIR-FPs has overcome several limitations of conventional FPs, such as photobleaching and slow chromophore maturation, while considerably enhancing imaging quality. Additionally, they increase imaging depth and speed, reduce phototoxicity, and improve resolution, representing considerable advancements for high-performance biological imaging.

2.5. GFP-like chromophore derivatives

The intrinsic GFP, HBI, serves as a foundational scaffold for the development of structurally similar small-molecule dyes. Its conjugated framework and the electronic delocalization between the phenolic hydroxyl and imidazolinone units are key to its environment-sensitive luminescent properties. Inspired by these characteristics, researchers have designed a series of chemically modified HBI derivatives that mimic fluorescence even in the absence of the native protein matrix.

3,5-difluoro-4-hydroxybenzylidenimidazolinone (DFHBI) exemplifies this approach, where the substitution of electron-withdrawing atoms like fluorine or chlorine on the phenyl ring alters the electron distribution and enhances resistance to photobleaching [77]. In 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), replacing the imidazolinone group with a thiazolinone unit changes the delocalization pathway, resulting in bathochromic shifts that bring the emission closer to that of red-emitting proteins [78]. BI-type compounds are further optimized through π-extension via vinyl bridges or the addition of rigidified aromatic moieties, which enhance both emission efficiency and structural stability [79].

Chemical diversification of the HBI backbone with heterocycles, such as pyridines, quinoxalines, or quinazoline derivatives, adds responsiveness to factors like pH, solvent polarity, or excitation wavelength. For example, LSFP-Yellow and LSFP-Red, two large Stokes shift fluorescent proteins (LSFPs), feature extended π-systems incorporating donor–acceptor motifs and are specifically designed for trace detection applications, such as forensic visualization of latent fingerprints [80]. These synthetic dyes not only replicate the core topology of GFP-like chromophores but also achieve fluorescence activation through entirely abiotic frameworks. Their small size, structural flexibility, and robust optical performance make them highly suitable for imaging, sensing, and molecular diagnostics in systems where genetic encoding is not practical.

3.1. Fluorophore-binding RNA aptamers

RNA, a key regulator in genetic processes, is closely associated with the pathogenesis and progression of various diseases. In studies of the dynamic mechanisms of RNA transcription, translation, transport, localization, and degradation, fluorescent RNA aptamers function similarly to GFP-tagged proteins, enabling low-background, in-situ, real-time imaging of target RNA through fluorescence-based assays [81,82]. Recently developed fluorescent RNA aptamers offer an optimized and efficient approach for labeling RNA. These aptamers are characterized by their ease of use, minimal interference with target RNA, and high signal-to-noise ratio. A key feature of these aptamers is their ability to noncovalently and reversibly bind non-fluorescent small molecules, thus activating their fluorescence [83]. This method prevents fluorescence bleaching and allows for fluorescence restoration. The interaction between small-molecule fluorophores and RNA aptamers follows a three-step kinetic process: binding, conformational changes, and dissociation. These steps occur in a cyclic manner, creating a dynamic loop in which small-molecule ligands bind to and dissociate from RNA aptamers, restoring fluorescence (Figure 3).

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Figure 3.

Selection and kinetic behavior of fluorescent RNA aptamers. (a) RNA aptamers are selected using SELEX to bind target molecules with high affinity and specificity. (b) Transient binding to small-molecule fluorophores enhances fluorescence, followed by photoisomerization-induced quenching and dissociation. The aptamer then binds a new fluorophore, enabling kinetic fluorescence cycling. Figure created with BioRender (https://BioRender.com).

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The first fluorescent RNA aptamer to mimic GFP, Spinach, specifically binds to the phenolic form of DFHBI [8]. This binding effectively prevents nonradiative decay by restricting the free rotation of internal bonds within the fluorophore, forming a Spinach-DFHBI fluorescent complex with an intensity comparable to that of traditional FPs [84-86]. Due to its resistance to photobleaching, Spinach exhibits fluorescence shortly after transcription when coupled with target RNA, enabling continuous, in-situ luminescence in live cell imaging [8]. Additionally, Spinach has been adapted into small-molecule sensors for real-time monitoring of transcriptional processes both ex vivo and in vivo [87-90] as well as tracking the dynamics of cellular metabolites [77,91,92]. This demonstrates excellent cellular compatibility, avoiding non-specific activation by endogenous cellular components, such as malachite green [93]. As the first RNA-based system to enable genetically encoded fluorescence and real-time visualization of RNA in live cells, Spinach overcomes key limitations of traditional RNA aptamers, including high cytotoxicity and incompatibility with live cell environments. This breakthrough paves the way for the development of future fluorescent RNA aptamers.

However, the thermal instability and propensity of Spinach to misfold can lead to reduced fluorescence intensity when fused with target RNA [95]. To address this, modifications were introduced to Spinach’s stem-loops 1 and 3, which are tolerant to base mutations and insertions, resulting in a variant known as Spinach2. This variant offers improved brightness and thermal stability, making it particularly useful for imaging low RNA concentrations [95,77,103]. In 2014, researchers targeted the third stem of the Spinach RNA aptamer, a functionally irrelevant yet conserved region, creating Split Spinach. While Split Spinach exhibits 20% lower fluorescence intensity than Spinach2, its development laid the groundwork for studying RNA assembly and RNA-RNA interactions [104]. A novel RNA fluorescent aptamer, Broccoli, was developed simultaneously. It represents a de novo optimization of Spinach, identified through a combination of SELEX and fluorescence-activated cell sorting (FACS) to screen aptamers expressed in E. coli, followed by further refinement using FACS-based directed evolution. As a result, Broccoli exhibits higher fluorescence intensity than Spinach2 and remains structurally stable, without misfolding, even in the presence of magnesium ions [9], making it suitable for imaging single mRNA molecules [105]. Additionally, Alam et al. utilized a three-way junction (3WJ) structure to divide Broccoli and incorporated the gene for mCherry, a red FP, into this system, facilitating a multistep fluorescence reaction [106].

Spinach and Broccoli aptamers emit green fluorescence, making them widely used in the development of RNA fluorescent probes. However, endogenous cellular molecules such as flavins and NADH also produce green background fluorescence, which can interfere with probe signals. Moreover, fluorescence intensity can fluctuate based on the metabolic state of these molecules [107]. To address this issue, researchers used SELEX-guided selection with DFHO, a structural analog of DsRed, as the ligand, resulting in the isolation of three fluorescent RNA aptamers: Corn, Orange Broccoli, and Red Broccoli [82]. These aptamers not only expand the spectral range of RNA-based fluorescence but also provide valuable insights into the role of intracellular metabolites in regulating biological pathways. Additionally, the inherently low background fluorescence in the red region for cells enhances their suitability for in vivo imaging applications [108,109], offering improved precision in intracellular imaging. While the Red Broccoli aptamer forms a red-emitting complex with DFHO in vitro, the fluorescence signal is insufficient for reliable detection in mammalian cells. To address this challenge, Li et al. designed a new fluorophore, 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzimidazole (OBI), which incorporates a benzimidazole substitution at the N1 position of DFHO. This structural modification enhances the conformational stability of the Red Broccoli aptamer and considerably boosts its photostability, enabling sensitive in vivo monitoring of S-adenosyl methionine (SAM) dynamics [92]. Notably, the fluorophore-interacting motifs of red and orange broccoli differ from those of Spinach by just a single nucleotide [78], indicating that the sensor engineering framework developed for Spinach can be easily adapted to these Broccoli variants, facilitating the creation of structurally analogous RNA-based imaging tools.

The yellow fluorescent aptamer Corn activates DFHO fluorescence through a G-quadruplex and dimeric structure, providing excellent photostability for long-term imaging, which is often essential for monitoring transcriptional processes in vivo [82]. While Corn requires dimerization to engage and activate DFHO fluorescence, this structural dependency limits its use for mRNA labeling and imaging in live cells. For these applications, a photostable RNA-fluorophore complex that functions in its monomeric form would be more suitable.

These aptamers consistently exhibit G-quadruplex structures [110], which are crucial for activating the luminescent properties of the chromophore (Figure 4a) [78,111]. Structural analysis of the Spinach aptamer–fluorophore complex showed that the fluorophore is securely positioned between a base triple, a G-quadruplex motif, and exposed guanine residues within the RNA scaffold [78] (Figure 4b). However, the structural stability of these G-quadruplexes in fluorescent RNA aptamers is highly sensitive to variations in cellular conditions, such as changes in metal ion concentrations and pH [112]. These environmental fluctuations can hinder the reliable tracking of target RNA species. Additionally, the inherent complexity of the G-quadruplex structure increases the risk of conformational misfolding within the aptamer scaffold [113].

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Figure 4.

DFHBI binding mechanism to G-quadruplex RNA aptamers. (a) Alignment of Spinach, 29-1, Broccoli, and their derivatives shows a conserved 32-nt core (dashed line) critical for DFHBI recognition through G-quadruplex formation. (b) Structural representation of DFHBI bound within the Spinach G-quadruplex; nucleotide colors match those in panel (a), emphasizing the correspondence between structure and function. Reprinted with permission from ref (78). Copyright [2019] American Chemical Society.

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Yang et al. utilized SELEX technology to identify RNA aptamers named Pepper, which demonstrate high affinity, remarkable stability, and considerable pH tolerance while exhibiting diverse spectral properties in their monomer form within living cells [97]. In contrast to G-quadruplex structures, Pepper aptamers bind to synthetic dye derivatives of hydrazone-cyanobenzene (HBC) through base-quadruplex and base-triple stacking interactions [114]. This binding mechanism enables in-situ labeling of various RNA species in eukaryotic cells, facilitating high-contrast imaging and overcoming key challenges associated with real-time RNA visualization in live-cell environments [98]. RNA sensors derived from Pepper have been engineered to quantify and visualize a wide range of intracellular targets, including metabolites, pharmaceutical compounds, proteins, and metal ions [115,116]. These sensors are also capable of detecting targeted RNA [117]. Moreover, RNA origami design strategies have been used to engineer Pepper and Broccoli aptamers into a novel FRET-compatible pair, allowing for precise three-dimensional spatial arrangement of the aptamer components [118]. This advancement provides a foundation for designing intracellular sensors and developing ratiometric biosensors [119].

Squash, a fluorescent aptamer that does not rely on G-quadruplex structures, was derived from a bacterial adenine riboswitch. It binds to DFHBI-1T or DFHO, emitting orange fluorescence [99]. Like Pepper, Squash operates as a monomer. Its riboswitch-based tertiary structure offers a stable framework that maintains the structural integrity and functional characteristics of biological RNA through evolutionary adaptation. As a result, Squash demonstrates improved photostability compared to Broccoli, a higher binding affinity for DFHO, and more efficient intracellular folding than Corn [100]. Peng et al. took advantage of the distinct emission wavelengths of Squash and Pepper to create a highly efficient dual-color orthogonal miLS imaging platform, utilizing the toehold-mediated mechanism [120]. This platform allows for precise imaging of specific miRNAs in live cells, offering substantial potential for diagnosing and treating diseases related to miRNA dysregulation [121]. LSFPs are commonly used for multicolor fluorescent labeling and live cell and protein imaging [122]. Similarly, the fluorescent molecule NBSI was designed to mimic the large Stokes shifts observed in red FPs and was utilized in SELEX technology to generate Clivias, a fluorescent RNA aptamer with a large Stokes shift [123]. This aptamer enhances fluorescence via an intermolecular proton transfer mechanism [124], enabling real-time, dual-emission RNA and genomic locus imaging in live cells with a single excitation source and supporting the detection of RNA-protein interactions.

3.2. Three-dimensional DNA structures

DNA naturally exists as a double helix, with its structural stability relying on hydrogen bonds between base pairs and base-stacking interactions on complementary strands. The functionality and biological activity of DNA are determined by its nucleotide sequence [125-127]. However, recent studies have uncovered additional biological functions of DNA beyond its traditional role in genetic information storage. DNA mimics, such as Lettuce, have shown the ability to form non-traditional three-dimensional structures through non-helical regions, mimicking the luminescence of FPs [11]. This discovery considerably broadens our understanding of DNA’s functional diversity and expands its conventional roles.

Lettuce is a DNA aptamer based on the small-molecule analog 4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one (DFHBI-1T), identified through SELEX. As the first known DNA analog of GFP, Lettuce is of substantial importance in molecular biology and fluorescence imaging [101]. Structural studies of the Lettuce–DFHBI-1T complex have revealed the molecular mechanisms by which DNA structures can mimic GFP-like fluorescence. The functional core of Lettuce adopts a four-way junction (4WJ) topology, forming a complex three-dimensional scaffold stabilized by extensive base stacking, hydrogen bond networks, and coordination with both mono- and di-valent metal ions. This structural configuration enables Lettuce to activate its bound fluorophores exclusively through its native folding pathway, without relying on RNA or protein co-factors [11]. The G-quadruplex Q2 region [128,129], located at the center of Lettuce, plays a critical role in fluorophore binding [11]. Due to its remarkable photostability and its utility as a gel-imaging marker, Lettuce DNA can be specifically detected even at low concentrations in highly complex nucleic acid samples [101].

The Split Spinach system, which uses ribonucleic acids to detect extracellular RNA, is susceptible to degradation by ribonucleases. To address this challenge, researchers developed a Split Lettuce sensor, specifically engineered to detect small amounts of viral RNA, including that of SARS-CoV-2 (Figure 5) [101]. Furthermore, a Lettuce sensor was combined with the AS1411 aptamer to create a DNA-based fluorescence sensor capable of accurately and selectively detecting Cu²⁺ both in vitro and in cellular environments [130].

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Figure 5.

Split lettuce aptamer function. (a) Schematic of the Split Lettuce sensor architecture, with modifiable regions indicated. (b) Different sensor pairs adopt varied three-way junction (3WJ) conformations, resulting in distinct fluorescence outputs. (c) Representative 3WJ configurations formed between target RNA (black) and two sensor strands (gray). (d) Sensor designs effective for one RNA site also retain activity across three additional RNA targets. (e) Fluorescence activation of the Split Lettuce system is dependent on RNA sequence. (f) Fluorescence intensity increases over time, reflecting the kinetics of aptamer reassembly. Adapted with permission from ref. [101]. Copyright (2022) American Chemical Society.

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Kanamori et al. innovatively engineered oligo-deoxynucleotides (ODNs) into triplex DNA architectures that replicate the chromophore-embedding function of the β-barrel in GFP. By tethering molecular rotor-type dyes to the C5 position of deoxyuridine, they created structurally responsive fluorescent probes whose emission characteristics are governed by triplex formation. This strategy provides a nucleic acid-based route to controllable fluorescence activation, facilitating sensitive detection of DNA structural transitions [131].

Building on the structural versatility of DNA, origami nanotechnology has enabled the precise spatial assembly of functional motifs with nanometer-scale accuracy [132]. This approach provides a framework for creating confined excitation environments, where fluorophores can be immobilized and oriented similarly to the steric restrictions imposed by the β-barrel fold of GFP [133]. Such configurations allow for precise control over both the photophysical properties of fluorophores and their interactions. For example, researchers employed a spatially directed strategy to arrange dye molecules within DNA-based scaffolds, investigating how geometric constraints influence energy transfer efficiency [134]. Through close-contact stacking and rigid placement, they enforced planarity and positional stability among dyes, thus recapitulating the physical confinement required for efficient excitation transfer [135]. The resulting FRET efficiencies were tunable in terms of both spatial separation and angular orientation, showing that DNA nanostructures can mimic directionally selective energy pathways typically reliant on protein frameworks [136]. The spatial programmability of DNA origami enables the precise three-dimensional organization of photonic components, facilitating the placement of multiple fluorophores, dynamic signal regulation, and seamless integration with logic-operable molecular circuitry [137]. This programmable framework supports the design of artificial fluorescence systems that mimic GFP-like behavior—specifically, structure-induced activation, spatial precision, and controllable emission—thus paving the way for nucleic acid-based optical devices in biosensing, imaging, and nanophotonic applications.

3.3. Chemically conjugated FPs

Peptide nanostructures designed to exhibit tunable and visible fluorescence, resembling GFP luminescence [102,138], have substantial potential for biomedical applications. These structures exhibit exceptional photostability, biocompatibility, and biodegradability [139-141] while circumventing the cytotoxicity concerns typically associated with quantum dots [17]. The inclusion of hydrophobic ferrocene (Fc) enhances the aggregation of active peptides [142], facilitating the synthesis of fluorescent nanopeptide complexes that simulate GFP emission.

Non-pathogenic polypeptide virus-like particles (NVPs) were engineered by co-assembling a nuclear localization signal (NLS) from simian virus 40 with the V3 loop of the human immunodeficiency virus (HIV), linked through a ferrocene–phenylalanine (Fc-FF) dipeptide [17] (Figure 6). When excited at 490 nm, these nanostructures emit intense green fluorescence at 520 nm. The resulting NVPs demonstrate excellent photostability and biocompatibility while maintaining the membrane-penetrating and nuclear-targeting capabilities conferred by the HIV V3 sequence [143] and the simian virus NLS peptide [144]. These features promote efficient cellular uptake and prolonged intracellular retention without causing cytotoxicity. Additionally, NVPs can encapsulate nucleic acids and facilitate the delivery of genetic material into target cells, enabling real-time tracking and monitoring of intracellular gene delivery and expression dynamics.

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Figure 6.

Assembly and functional evaluation of fluorescent nanopeptides. (a) Fc-FF-NLS and Fc-FF-V3 form green-emitting nanoparticles through self-assembly and photoactivation. (b, c) atomic force microscopy (AFM) images showing peptides in monomeric form (b) and nanoparticle form (c). (d) AFM visualization of NVP@OLD hybrid structures. (e) Proposed pathways for fluorescence activation. (f) Photostability assessment under continuous illumination. (g) Cytotoxicity evaluation in CD4/CCR5+ Magi cells using an MTT assay. (h) Long-term cell imaging using NVPs (scale bar: 23 μm). (i, j) NVPs support nucleic acid encapsulation and enable tracking of gene delivery (scale bar: 9 μm). Adapted with permission from ref. [17]. Copyright (2019) American Chemical Society.

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However, assembling NVPs under non-physiological conditions, such as low temperature [145], organic solvents [146], and high-temperature processing [147], presents challenges for their use in biological imaging applications. To overcome these limitations, researchers modified tyrosine residues in the GFP chromophore with Fc, resulting in the creation of ferrocene–tyrosine fluorescent polypeptide (Fc-YY) nanoparticles [102]. The hydrophobic Fc moiety facilitates the co-assembly process, and under physiological conditions, Fc-YY nanoparticles exhibit green fluorescence, mirroring the optical properties of GFP and enabling stable cellular imaging over several days.

Oligopeptides, known for their inherent biocompatibility and photostability, are ideal candidates for the development of FP nanoparticles, particularly those derived from histidine-rich oligopeptides [138]. By harnessing the hydrogen bonding and π-π interactions of these peptides, researchers have successfully formed nanoclusters that subsequently self-organize into layered nanoassemblies. These assemblies exhibit tunable fluorescence, ranging from blue to orange under UV excitation, with fluorescence intensity regulated by histidine concentration. The dynamic chromogenic properties of these histidine-rich peptides, which can exhibit multicolor fluorescence in response to varying intracellular histidine levels, make them highly promising for biomedical applications [148].

Currently, Fc-modified histidine dipeptide nanoassemblies are employed as quantitative ratiometric biosensors, emitting bicolor fluorescence that shows a linear correlation with peptide concentration, driven by hydrogen bonds and aromatic interactions. This quantitative capability for biomolecular analysis is comparable to that of FPs [149]. Furthermore, by using 12 different Fc-modified, non-fluorescent oligopeptides (Fc-HHH) as building blocks, FP nanostructures are formed through aggregation. The aggregation-induced emission arises from the restricted intramolecular movement of specific amino acids—phenylalanine, tyrosine, tryptophan, and histidine—resulting in a full visible-light spectrum, a phenomenon referred to as the peptide-based nano-rainbow kit (PRK) [138] (Figure 7). PRK is particularly well suited for monitoring biomolecules or individual virus particles, precisely localizing biological structures, and serving as an intracellular biosensor [150,151]. The photophysical adaptability and functional versatility of PRK can be further enhanced through strategies such as extending peptide backbones, optimizing metal ion coordination environments, and introducing expanded π-conjugation frameworks [138].

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Figure 7.

Design and evaluation of PRK FPs. (a) GFP-inspired design strategy of PRK. (b) Structures and corresponding fluorescence hues of PRK peptides. (c) Coarse-grained simulation of CPD-HHH self-assembly. (d) Atomistic modeling snapshots from 0 to 2000 ns. (e) Fluorescence imaging of HeLa cells labeled with PRK (scale bar: 23 μm). Adapted with permission from ref. [136]. Copyright (2020) American Chemical Society.

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3.4. De novo designed protein

With advances in computational protein engineering and structural prediction techniques, it is now increasingly feasible to design synthetic proteins that exhibit programmable fluorescence. Unlike naturally evolved FPs, which emerge through iterative evolutionary selection, these artificially constructed proteins are generated ab initio, without relying on pre-existing templates. Their design utilizes physics-informed modeling frameworks and algorithmic platforms like Rosetta, allowing for the specification of novel folds and precise fluorophore-interacting microenvironments [26]. While these constructs do not share evolutionary ancestry with canonical proteins like GFP, their design concept is functionally analogous: immobilizing small molecule fluorogens within rigid scaffolds to minimize vibrational relaxation and enhance photonic emission.

A notable example of this approach comes from David Baker’s laboratory, where they reported the de novo design of a β-barrel protein that selectively accommodates and activates DFHBI, a GFP-mimetic dye [26]. The engineered protein encapsulates the fluorogen within a structurally rigid cavity, stabilized through hydrophobic complementarity and hydrogen bonding. Although the quantum yield is lower than that of natural FPs, this achievement demonstrates the feasibility of designing complex protein folds that enable functional optical readouts.

Further advancements have led to the development of compact alternatives to the β-barrel motif. For example, mini fluorogen-activating proteins (mFAPs) feature compact structures adorned with aromatic residues [152,153], creating low-entropy pockets that engage fluorogens through π-stacking and hydrophobic interactions. In their unbound state, the dyes dissipate energy via internal rotation, and binding to the mFAP scaffold halts this motion, thus triggering fluorescence. The small size of these proteins makes them well suited for integration into cell-compatible imaging, nanoscale biosensing, and single-molecule studies. However, variability in the electrostatic properties of the binding cleft may contribute to nonspecific background signals.

Subsequent iterations of this concept include the Fluorescence-activating and absorption-shifting tag (FAST) (and nanoFAST) series of small fluorogen-activating proteins, which employ structurally minimal yet effective folds to bind dyes like DFHO with high selectivity. These designs combine β-strands and α-helices to form reversible binding pockets, generating rapid and tunable fluorescence upon ligand binding [154]. Their small size and reversible activation make them particularly advantageous for high-resolution live-cell microscopy, where they enable orthogonal multiplexing alongside GFP derivatives [155]. Additionally, their favorable photophysics and tunable properties have made them ideal for use in dynamically responsive biosensors, capable of accurately reporting intracellular fluctuations in redox balance, metabolite concentrations, and local signaling states [156].

Beyond molecular tagging, these synthetic constructs are being adapted for use in engineered biosensing circuits. By designing ligand-responsive binding pockets, they can function as “switchable” fluorescent components with high on/off contrast. This capability enables their application in microfluidic diagnostics, surface-tethered assays, and implantable sensor devices, where spatial and temporal precision is essential. Their orthogonality to endogenous components further enhances their suitability for longitudinal monitoring in live tissues and 3D culture systems.

A distinct innovation trajectory is represented by esmGFP, an FP entirely constructed through machine learning-driven sequence generation [157]. Using the ESM-3 language model, researchers identified sequence variants capable of fluorescing, even in the absence of recognizable structural domains typically found in known FPs. The photophysical mechanism is still under investigation, potentially involving cryptic chromophore formation or the emergence of novel cavity architecture. Parallel advances from ESMFold have demonstrated that accurate structural prediction from primary sequences alone is now achievable, enabling faster design cycles and broader sequence exploration [158]. These breakthroughs open new possibilities for designing function-specific fluorogenic proteins, optimized for detecting cellular ionic fluxes, pH microdomains, or enzyme activities under physiological conditions.

Taken together, these efforts represent a transformative shift in fluorescent system design from repurposing naturally occurring scaffolds to creating tailored, algorithmically derived photoreactive modules. With their smaller steric footprint, versatile emission profiles, and minimal crosstalk with cellular components, these proteins offer distinct advantages for synthetic biological devices, subcellular visualization, and in-situ biosignal detection. As fluorogen chemistry continues to evolve and AI-guided engineering advances, these next-generation constructs are set to play a pivotal role in programmable diagnostics, advanced imaging, and optochemical control strategies. Looking ahead, key areas for development include expanding their spectral range into the near-infrared, engineering allosteric fluorescence control, and integrating them into wearable or implantable biosensor platforms for clinical applications.