RNA Mango-based sensors for lead†

Annyesha Biswas^a and Saurja DasGupta*^abc
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. E-mail: sdasgupta@nd.edu
^bDepartment of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
^cBerthiaume Institute for Precision Health, University of Notre Dame, Notre Dame, IN 46556, USA

First published on 27th June 2025

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Introduction

Heavy metal contamination is a persistent global threat to environmental and human health. Among these metals, lead (Pb²⁺) is of particular concern due to its high toxicity even at low exposure levels. Industrial processes such as the mining and smelting of lead ores,¹ the production of leaded gasoline,² and the manufacturing of lead-acid batteries³ and lead-containing pigments⁴ release large amounts of lead into the environment. Importantly, chronic exposure to low amounts of lead, primarily through contaminated drinking water,⁵ industrial waste,⁶ and deteriorating infrastructure such as lead-based pipes,⁷ has been linked to cognitive deficits,⁸ developmental disorders,⁹ cardiovascular diseases,¹⁰ and kidney dysfunction.¹¹ Pb²⁺ toxicity poses a heightened risk to children, where exposure is associated with neurodevelopmental impairments, behavioral disorders, and reduced attention spans.^12,13 Given these health risks, regulatory agencies such as the U.S. Environmental Protection Agency (EPA) and the World Health Organization (WHO) have set stringent limits for lead in drinking water at 15 parts per billion (72 nM) and 10 parts per billion (46 nM), respectively.^14,15 Ensuring compliance with these regulations requires sensitive and reliable detection methods.

Traditional analytical techniques for Pb²⁺ detection include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, anodic stripping voltammetry, and X-ray fluorescence spectroscopy.¹⁶ These methods allow highly sensitive Pb²⁺ detection; however, the requirements for expensive and elaborate instrumentation, trained personnel, and complex sample preparation make them less suited for rapid, on-site monitoring. Organic scaffolds, like small molecule chelators, macrocycles, or even metal–organic frameworks (MOFs), have been used for fluorescence-based and colorimetric detection of Pb²⁺.^17–19 Although these chemical sensors are capable of detecting Pb²⁺ without sophisticated instrumentation, they are not sufficiently selective and often fail to detect trace amounts of Pb²⁺.

To address these limitations, there is increasing interest in developing biosensors that offer rapid, cost-effective, and portable lead detection with minimal technical expertise. Biosensors generally outperform chemical sensors in selectivity and sensitivity metrics, presumably due to more sophisticated molecular recognition by biomolecules.^20,21 Nucleic acid-based biosensors detect Pb²⁺ by leveraging structural changes induced by Pb²⁺ binding.^22,23 For example, Pb²⁺ binding to RNA-cleaving DNAzymes induce the formation of their catalytic conformation, which results in the cleavage of an RNA strand.²⁴ Pb²⁺-induced cleavage of a fluorescently-labeled RNA substrate spatially separates the fluorophore on the RNA from the quencher on the DNAzyme, generating a fluorescence ‘On’ signal. A similar strategy can be used to create colorimetric DNAzyme-based sensors by conjugating them to gold nanoparticles.²⁵ G-quadruplex-based biosensors exploit the Pb²⁺-induced folding of guanine-rich sequences into a G-quadruplex. G-quadruplexes are typically formed in the presence of monovalent cations like Na⁺ and K⁺.²² Pb²⁺, due to its similar ionic radius to K⁺ and twice the charge, facilitates the formation of a stable G-quadruplex structure at lower concentrations. G-quadruplex-based sensors exploit the structural change from single-stranded to a G-quadruplex in different ways.²⁶ Pb²⁺-induced quadruplex formation may bring the two DNA termini, modified with a fluorophore and quencher, into close proximity, resulting in a fluorescence ‘Off’ signal.²⁷ Certain G-quadruplex sequences exhibit peroxidase activity upon binding to hemin and appropriate reactants like ABTS, generating colored, chemiluminescent, or fluorescent products.²⁸ Therefore, the catalytic activity of these quadruplex-hemin DNAzymes has also been used as a Pb²⁺ sensing platform. DNA-based electrochemical sensors convert G-quadruplex formation into an electrochemical signal, which is detected by techniques such as cyclic voltammetry.²⁹ However, the requirement for nucleic acid modification with fluorophores/quenchers or nanoparticles, pH-sensitive chemical reactions, or expensive electrochemical assemblies poses significant challenges to the practical application of these biosensors.

A new strategy that leveraged Pb²⁺-induced G-quadruplex formation in the fluorogenic RNA aptamer, Spinach, generated the first RNA-based biosensor for Pb²⁺.³⁰ Stabilization of the Spinach G-quadruplex by trace amounts of Pb²⁺ allows its cognate dye, DFHBI, to bind to this platform, activating strong and stable green fluorescence. Spinach demonstrated moderate binding affinity for Pb²⁺ (K_D ∼ 1.3 μM) but high sensitivity, with a limit of detection (LOD) of 6 nM. This system consists of just the RNA aptamer and its cognate dye, where the fluorescence signal results from non-covalent dye binding. Therefore, fluorogenic RNA aptamers offer a simple and efficient platform for Pb²⁺ detection, addressing key limitations related to covalent modifications of nucleic acids with dyes or nanoparticles, optimal reaction conditions for colorimetric or chemiluminescence detection, and elaborate electrochemical assemblies. Despite the creation of numerous G-quadruplex-containing fluorogenic RNA aptamers since its development, the Spinach sensor remained the only RNA-based biosensor for Pb²⁺. In this study, we expand the repertoire of RNA-based Pb²⁺ sensors by evaluating alternative fluorogenic RNA aptamers for enhanced binding affinity to Pb²⁺, increased sensitivity, and improved selectivity. We identify the RNA Mango aptamer as an excellent candidate for creating Pb²⁺ sensors and describe its Pb²⁺-sensing properties in combination with three dyes: TO1-biotin and TO3-biotin, the cognate dyes for this aptamer, and thioflavin-T (ThT), a sequence agnostic but G-quadruplex-specific dye.^31–34 A detailed characterization of the fluorescence properties of the Mango aptamer with these dyes in the presence of Pb²⁺ allowed us to develop the most sensitive RNA-based Pb²⁺ sensors to date.

Results and discussion

RNA Mango exhibits strong fluorescence in the presence of Pb²⁺

Since the development of the Spinach sensor, other G-quadruplex-containing fluorogenic RNA aptamers such as Broccoli, Mango, Peach, and Beetroot have been artificially evolved (Fig. S1†).^35–38 The Broccoli (Fig. S1A†) and Beetroot (Fig. S1B†) aptamers were isolated from random RNA libraries by virtue of their ability to bind to DFHBI-1T and DFHO and exhibit green and yellow fluorescence, respectively (Table S1†).^35,36 The Mango aptamer (Fig. S1C†) was similarly obtained for binding to thiazole orange derivatives, namely TO1-biotin and TO3-biotin.³⁹ Various RNA Mango aptamers have been reported in the literature – all of which bind to their cognate dyes with nanomolar affinities. Mango III exhibits the brightest fluorescence among the existing Mango aptamers (Mango aptamers I–IV),⁴⁰ and the A10U mutant of Mango III (MangoIII_A10U) was reported to show a higher quantum yield and 18% higher fluorescence enhancement over wild-type Mango III.³⁸ Given our goal of developing a fluorescence sensor for Pb²⁺, we selected the Mango III_A10U aptamer due to its superior fluorescence properties. The availability of a high-resolution crystal structure of Mango III_A10U during our screening process afforded opportunities for engineering a truncated version of this aptamer (Fig. S1C†).³⁸ Peach, an aptamer related to Mango, was selected for binding to TO3-biotin.³⁷ The G-quadruplex motif in each aptamer, stabilized by 100 mM K⁺ in the folding buffer, serves as the dye binding platform and is consequently key to the aptamer's fluorescence properties. To assess whether these G-quadruplex-containing RNA aptamers can be used to detect Pb²⁺, we examined the fluorescence properties of Broccoli, Mango III_A10U (henceforth as Mango), Peach, and Beetroot in the presence of their respective dyes, both in the absence and presence of 1 μM Pb²⁺ (Fig. 1 and S2†).

Although all four aptamers use G-quadruplexes for binding to their cognate dyes, only RNA Mango in combination with TO1-biotin exhibited a significant fluorescence enhancement (∼8-fold) in the presence of 1 μM Pb²⁺ (Fig. S2A†). The Peach/TO3-biotin combination also showed fluorescence enhancement upon the addition of Pb²⁺; however, this enhancement was less pronounced (3-fold) with a less intense signal than Mango/TO1-biotin (Fig. S2B†). No noticeable enhancement in fluorescence was observed for the Broccoli/DFHBI-1T and Beetroot/DFHO aptamer-dye pairs (Fig. S2C and S2D†). This could be due to the relatively lower affinity of Broccoli and Beetroot for their cognate dyes DFHBI-1T (K_D ∼ 0.36 μM) and DFHO (K_D ∼ 2.6 μM) compared to the low nanomolar affinities of Mango and Peach to their dyes in ∼100 mM K⁺.^35–37 Despite the lower affinity for their dyes, the poor fluorescence enhancement observed in Broccoli and Beetroot is unexpected and may reflect the differences in their dye binding affinities in the presence of 1 μM Pb²⁺. Fluorescence enhancement exhibited by Mango/TO1-biotin in the presence of 1 μM Pb²⁺ was 33-fold, 308-fold, and 1449-fold higher relative to Peach/TO3-biotin, Broccoli/DFHBI-1T, and Beetroot/DFHO, respectively (Fig. 2A and B). These results suggested that, among the aptamers tested, Mango was the most promising candidate for a Pb²⁺ sensor.

As Mango showed the highest increase in fluorescence in the presence of Pb²⁺, we studied its fluorescence properties with TO3-biotin and ThT, dyes that have been previously reported to bind Mango in addition to TO1-biotin.^38,40 While TO3-biotin is a cognate dye of the Mango and Peach aptamers, ThT exhibits green fluorescence upon specifically binding to G-quadruplex structures in a context-independent manner (Table S1†).^31–34 Mango showed fluorescence enhancements of ∼2-fold and ∼5-fold in the presence of TO3-biotin and ThT, respectively, upon Pb²⁺ addition (Fig. 2C and D). This ability of Mango to exhibit fluorescence enhancements with all three dyes in the presence of just 1 μM Pb²⁺ highlights its robustness and versatility as a potential Pb²⁺ sensor.

Pb²⁺-induced fluorescence activation of RNA Mango is dependent on G-quadruplex stabilization

Stabilization of the G-quadruplex motif in Mango is central to its fluorescence activation by Pb²⁺ as this motif serves as the binding platform for the dye. RNA G-quadruplexes usually exist in the parallel topology and are characterized by a prominent positive peak at ∼260 nm and a prominent negative peak at ∼240 nm.⁴¹ We observed an enhancement of the positive peak at 263 nm upon the addition of 1 μM Pb²⁺ to Mango in the absence of any dye, which further increased in intensity in the presence of 10 μM Pb²⁺ (Fig. 3A). This Pb²⁺-induced increase in CD intensity at 263 nm was accompanied by a decrease in intensity at ∼240 nm. These results provide direct evidence of the Pb²⁺-induced stabilization of the G-quadruplex motif in Mango that is independent of dye binding.

To further investigate the effect of Pb²⁺-induced G-quadruplex formation on the fluorescence properties of Mango, we mutated guanine residues critical for quadruplex formation to adenines (Fig. 3B and Table S2†). Mutating these key guanine residues in the G-quadruplex resulted in a near-complete loss of fluorescence for TO1-biotin and ThT (Fig. 3C and E). Although significantly reduced, some fluorescence was detected in the quadruplex mutants of Mango when bound to TO3-biotin (Fig. 3D), likely due to the higher intrinsic fluorescence of TO3-biotin in buffer. Collectively, these results confirm that Pb²⁺-induced fluorescence activation of Mango is dependent on G-quadruplex formation.

RNA Mango is a sensitive Pb²⁺ sensor

An ideal Pb²⁺ sensor should exhibit high sensitivity with a limit of detection (LOD) in the low nanomolar range. To evaluate its sensitivity, we investigated Mango fluorescence with TO1-biotin, TO3-biotin, or ThT in response to increasing concentrations of Pb²⁺. The binding affinity of Mango to Pb²⁺ in the presence of each dye was determined by fitting the data to the Hill equation (Fig. 4A–C). The apparent K_D values for Pb²⁺ binding with Mango in the presence of TO1-biotin, TO3-biotin, and ThT dyes were calculated as 113 ± 31 nM, 295 ± 52 nM, and 157 ± 32 nM, respectively (Fig. 4D). These results revealed that sensors created with Mango/TO1-biotin, Mango/TO3-biotin, and Mango/ThT bind Pb²⁺ with 12-fold, 4-fold, and 8-fold greater affinities than the Spinach sensor (K_D ∼ 1.3 μM).³⁰ LOD of the Mango lead sensor with TO1-biotin, TO3-biotin, and ThT were calculated as 2 nM, 16 nM, and 4 nM (Fig. 4E and S3†), respectively (based on the 3σ/slope method, where σ is the standard deviation of the blank). This is consistent with the trend in their binding affinities for Pb²⁺. Sensors prepared with Mango/TO1-biotin and Mango/ThT are the most sensitive RNA-based Pb²⁺ sensors reported to date. Importantly, the LOD values for all three Mango-based lead sensors are significantly lower than the acceptable limits for Pb²⁺ in drinking water.

The increase in the fluorescence signal in response to increasing concentrations of Pb²⁺ was directly imaged for all three Mango-based sensors (Fig. S4†). While the Mango/TO1-biotin and Mango/ThT sensors exhibited a clear, concentration-dependent enhancement of green fluorescence upon Pb²⁺ addition with a negligible signal without Pb²⁺, the red fluorescence enhancement in the Mango/TO3-biotin sensor was less discernible due to the high intrinsic fluorescence of the TO3-biotin dye. Although Pb²⁺-induced fluorescence enhancement in the Mango/TO3-biotin sensor can be clearly measured by fluorimetry, its inability to provide a distinct visual readout of this enhancement prevents its use as a point-of-care lead sensor in its current form. In contrast, the ease of Pb²⁺ detection using the Mango/TO1-biotin and Mango/ThT combinations makes them attractive candidates for cheap, easy-to-use, point-of-care sensors for lead.

In addition to the sensors’ high affinity for Pb²⁺, the strong fluorescence signal in the presence of a trace amount of Pb²⁺ could be due to tighter binding of the dye. To assess the affinity of RNA Mango to the three dyes in the presence of 1 μM Pb²⁺, we performed fluorescence titrations in the presence of increasing concentrations of TO1-biotin, TO3-biotin, and ThT (Fig. S5A–C†). The apparent K_D values for dye binding were determined as 39 ± 8 nM for TO1-biotin (Fig. S5A and D†), 792 ± 72 nM for TO3-biotin (Fig. S5B and D†), and 193 ± 27 nM for ThT (Fig. S5C and D†). The highest affinity of Mango to TO1 is consistent with the highest sensitivity and Pb²⁺ affinity exhibited by the Mango/TO1-biotin sensor. In comparison, the apparent K_D value for DFHBI binding for the Spinach sensor in the presence of 10 μM Pb²⁺ was determined to be ∼1 μM.³⁰ Therefore, Mango-based lead sensors bind to their dyes with at least 1.4–28.5-fold greater affinity than the Spinach sensor. Consequently, Mango-based lead sensors require lower dye concentrations for Pb²⁺ detection.

RNA Mango is a selective Pb²⁺ sensor

To investigate the selectivity of Mango sensors for Pb²⁺ detection, we measured their fluorescence in the presence of thirteen other metal ions that may be encountered in environmentally relevant samples (Fig. 5). Pb²⁺-specific fluorescence signals could be detected visually for the Mango/TO1-biotin and Mango/ThT sensors; however, this was not possible for the Mango/TO3-biotin sensor, which showed a high background signal due to the intrinsic fluorescence of the TO3-biotin dye (Fig. S6†). In fluorimetric experiments, we observed strong fluorescence enhancement only upon Pb²⁺ addition with all three dyes; however, weak signals were detected in the presence of Ca²⁺ and K⁺ with TO1-biotin (Fig. 5A and B) and TO3-biotin (Fig. 5C and D). In contrast, the Mango/ThT sensor showed weak but detectable fluorescence only when Ca²⁺ was present (Fig. 5E and F). These results are consistent with reports of G-quadruplex stabilization by Ca²⁺ and K⁺, with the lower signals reflective of much weaker binding than Pb²⁺.³⁰ The Mango/TO1-biotin sensor exhibited ∼20-fold and ∼25-fold higher signals for Pb²⁺ over K⁺ and Ca²⁺, respectively (Fig. 5B), whereas the Mango/TO3-biotin sensor showed an ∼10-fold higher signal for Pb²⁺ relative to both Ca²⁺ and K⁺ (Fig. 5D). In contrast, the Mango/ThT sensor exhibited a 53-fold and 142-fold higher signal for Pb²⁺ compared to Ca²⁺ and K⁺, respectively (Fig. 5F). The superior selectivity for Pb²⁺, combined with a low limit of detection, makes Mango/ThT the best Mango-based lead sensor. The combined attributes of high binding affinity, excellent sensitivity, and strong selectivity for Pb²⁺ detection underscore the potential of Mango-based sensors as robust tools for the quantitative detection of Pb²⁺ at low concentrations.

RNA Mango detects Pb²⁺ in tap water

A reliable sensor must demonstrate functionality in practical, real-world applications. We tested the applicability of the Mango lead sensors for detecting Pb²⁺ in tap water. RNA Mango generated fluorescence within minutes of exposure to Pb²⁺ and remained stable in tap water containing 1 μM Pb²⁺ for at least 24 hours – the longest duration tested (Fig. S7†). Encouraged by this observation, we assessed the performance of the Mango/TO1-biotin and Mango/ThT sensors by spiking tap water samples with different concentrations of Pb²⁺. We observed an increase in the fluorescence signal with Pb²⁺ concentrations ranging from 5 nM to 5 μM for TO1-biotin (Fig. 6A) and ThT (Fig. 6B), indicating that the Mango sensor can detect Pb²⁺ in tap water. The increase in the fluorescence signal observed within minutes after the addition of Pb²⁺-spiked tap water and the stability of the Mango sensor in tap water demonstrate the potential applicability of our sensor in real-world samples. However, both Mango sensors showed modest background fluorescence in tap water samples, even in the absence of Pb²⁺. High concentrations of Ca²⁺ in the hard water samples may interfere with Pb²⁺ detection by Mango sensors; however, this can be remedied by several approaches, as discussed in the next section.

Conclusions

In this work, we created a new class of fluorescence biosensors for detecting Pb²⁺ based on the RNA Mango aptamer, thus expanding the analytical applications of RNA in heavy metal sensing. Notably, our sensors demonstrate excellent sensitivity with limits of detection (2–16 nM) significantly below the permissible level of Pb²⁺ in drinking water (72 nM). The Mango sensors are able to discriminate between the Pb²⁺ and other metal ions, showing only a weak signal with K⁺ and Ca²⁺. The Mango/ThT sensor emerged as the most promising candidate exhibiting high affinity for its analyte, Pb²⁺ and its dye, ThT in the presence of Pb²⁺, and showing the highest selectivity among the Mango sensors. The versatility of Mango-based lead sensors is highlighted by their compatibility with dyes spanning different spectral regions, including TO1-biotin and ThT in the green and TO3-biotin in the red regions of the spectra, which allows users to select the dye best suited to their needs. Additionally, to detect lead, one simply needs to add the sample to the RNA/dye solution, which eliminates the need for complex chemical reactions or sophisticated sensor assemblies. Fluorescence signal is observed in the presence of Pb²⁺ within minutes by simply shining light from a handheld UV lamp. These features make Mango lead sensors attractive for on-site detection of Pb²⁺.

However, to enable broad applicability in point-of-care devices, we are currently optimizing the concentrations of the Mango RNA aptamer and its fluorogenic dyes to reduce responsiveness to Ca²⁺, which, if present in real-world samples, may trigger weak false positives. Alternatively, water samples may be pre-treated with NH₄OH to precipitate Ca²⁺ as Ca(OH)₂, which can then be removed via filtration.⁴² A subsequent pH adjustment to ∼pH 8 with HCl will allow Mango sensors to function as intended. As Ca²⁺ is primarily present as Ca(HCO₃)₂ in hard water, boiling water samples will remove dissolved Ca²⁺ as CaCO₃, allowing Pb²⁺ detection by Mango sensors.⁴³ Another consideration for on-site detection is the potential to incorporate Mango sensors into inexpensive and portable point-of-care analytical devices. Toward this goal, we are developing paper-based analytical devices (PADs) and microfluidic chips for Pb²⁺ detection that use the Mango sensors reported in this work.

Experimental

Materials

Tris-HCl (pH 8) buffer was purchased from Invitrogen. Metal salts Pb(NO₃)₂·3H₂O, AgNO₃, CdCl₂·H₂O, CuSO₄·5H₂O, FeSO₄·7H₂O, NiNO₃·6H₂O, ZnCl₂, HgNO₃·H₂O, and Mn (CH₃COO)₂·4H₂O were purchased from Sigma-Aldrich (ACS grade). MgCl₂, NaCl, KCl, and CaCl₂ were purchased as filtered solutions from Invitrogen. Dyes DFHBI, DFHBI-1T, and DFHO were purchased from Lucerna Technologies. TO1-biotin and TO3-biotin were purchased from Applied Biological Materials Inc. (abm) and thioflavin-T (ThT) was purchased from Millipore Sigma. The details of all the dyes used in this work are listed in ESI Table S1.† The RNA oligonucleotides used in this work are listed in ESI Table S2† and were purchased from Integrated DNA Technologies (IDT).

Methods

Author contributions

A. B. and S. D. designed research; A. B. performed research; A. B. and S. D. analyzed the data; A. B. and S. D. wrote the paper.

Conflicts of interest

The authors declare that a provisional patent application has been filed related to the work described in this manuscript.

Data availability

Additional supporting data for this article have been included in the ESI.† This includes (1) chemical structures of the dyes and the sequences of the oligonucleotides used in this work, (2) secondary structures of the fluorogenic RNA aptamers tested and their fluorescence spectra with their corresponding dyes in the absence and presence of Pb²⁺, (3) sensitivity plots of the Mango sensor with TO1-biotin, TO3-biotin and ThT for Pb²⁺, (4) images of the Mango lead sensors with increasing concentrations of Pb²⁺ and different metal ions, (5) binding curves for measuring the affinity of TO1-biotin, TO3-biotin, and ThT to RNA Mango in the presence of 1 μM Pb²⁺, and (6) a denaturing PAGE gel to demonstrate the stability of the Mango lead sensor in tap water in the presence of 1 μM Pb²⁺.

Acknowledgements

We thank Professor Bradley Smith and Hailey Salaberry for providing access to their lab's fluorimeter. We thank the members of the DasGupta Lab for their valuable feedback on the manuscript. We thank Professor Marya Lieberman and Vikrant Jandev for sharing the salts used in this study. We thank high school summer students Areej Arif, Jennifer Yang, and Ishita Awasthi for their help during the initial phase of the project. We thank the Biophysics Core Facility for providing the CD spectrometer and Azure 600 imager. CD analyses were performed at the Notre Dame Biophysics Instrumentation Core Facility with support from NIH grant S10OD036273. This work was supported by the University of Notre Dame Start Up funds to S. D.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5ob00765h

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