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. 2024 Apr 9;13(4):1093–1099. doi: 10.1021/acssynbio.4c00009

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

Madeline M Mumbleau †, Fabienne Chevance ‡, Kelly Hughes ‡, Ming C Hammond †,*
PMCID: PMC11037261  PMID: 38593047

Abstract

graphic file with name sb4c00009_0006.jpg

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC–Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30–tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

Keywords: RNA aptamer, fluorescent RNA, fluorescence microscopy, flow cytometry, transcriptional reporter

Introduction

RNA is a highly dynamic and structurally diverse biomolecule that can be engineered for programming gene expression and cellular function within the field of synthetic biology.1−3 Among functional RNA elements, fluorogenic aptamers are an emerging strategy for tagging and imaging other RNAs and for monitoring cellular processes, as these aptamers emit fluorescence when bound to their cognate fluorophores, mimicking the properties of fluorescent proteins.4−8 To date, researchers have leveraged the versatility of fluorogenic aptamers for diverse applications, including tagging RNAs, imaging RNAs, assessing genetic circuits, and engineering fluorescent biosensors for small molecule analytes through fusion with riboswitch aptamers.9−17

Recently, the fluorogenic Pepper aptamer was developed, which is small in size (43 nt) and has low nanomolar binding affinities to cognate dyes (Figure 1A,B), enhanced thermostability and photostability, and robust fluorescence in vivo.18 A notable feature of the Pepper aptamer is that it can bind to eight different HBC dye analogs to give fluorescence emission across the visible spectrum, ranging from cyan to red.18 Although some studies have demonstrated the robustness of this aptamer in vitro and in eukaryotic cells,18−23 its activity in different bacteria remains largely unexplored. Only recently, a series of Pepper-based biosensors have been tested in E. coli.24 In fact, most fluorogenic aptamers have not been tested or applied outside of E. coli, even though many other bacterial species are applied industrially to produce chemicals, enzymes, and other biosynthetic products. There is also major interest in studying gene expression and other biological pathways in both pathogenic and commensal bacteria for health applications, from antibiotic development to probiotics to cancer targeting.

Figure 1.

Figure 1

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Assessment of the Pepper aptamer in three bacterial species. (A) Scheme of the HBC–Pepper complex. Fluorogenic HBC dyes become fluorescent when bound by the Pepper aptamer. (B) Structure of HBC dyes, HBC514 and HBC530. (C–E) Flow cytometry data of E. coli, B. subtilis, and S. Typhimurium cells expressing tRNA scaffolded Pepper with all HBC dye analogs. The emission filters used to analyze each HBC dye are indicated. Mean fluorescence intensity (MFI) values were determined by analyzing 30 000 cells per sample after 1 h of incubation with HBC dye (200 nM). Data shown are the average with a standard deviation of three biological replicates.

Here, we evaluate and compare the Pepper aptamer and the eight different HBC fluorophores for live cell imaging in E. coli, Salmonella Typhimurium, and Bacillus subtilis, two Gram-negative and one Gram-positive bacterial species. E. coli is a workhorse chassis for synthetic biology tool development, while pathogenic strains produce Shiga toxins and other virulence factors that lead to severe symptoms such as food poisoning, sepsis, meningitis, or urinary tract infections. B. subtilis has been applied industrially to produce chemicals and enzymes. S. Typhimurium is a pathogen that can cause foodborne illness and typhoid fever in humans. In addition, we show that placement of the aptamer in an optimal scaffold can dramatically affect the cellular fluorescence signal, with up to 199-fold greater fluorescence than the aptamer alone. Furthermore, the optimal scaffold also can display two different aptamers with orthogonal fluorescence signals for simultaneous two-color imaging.

Results

Assessment of HBC Fluorophores in Different Bacteria

We initially assessed the HBC–Pepper system with all eight HBC fluorogenic dyes in E. coli, B. subtilis, and S. Typhimurium. For in vivo studies, the Pepper aptamer was placed into a tRNA scaffold, which has been shown to promote better aptamer folding and improve the stability of RNA constructs against RNases within cells.25−27 Through quantitative analysis by flow cytometry, we observed that six of the eight HBC dyes could permeate into all three bacterial species and fluoresce upon binding to the Pepper aptamer (Figure 1C,D), whereas HBC508 and HBC599 consistently resulted in low cellular fluorescence.

To test whether the observed low fluorescence was due to suboptimal emission filters in the flow cytometer, bulk cellular fluorescence was analyzed for the three lowest dyes, HBC497, HBC508, and HBC599, in a fluorescence plate reader outfitted with a monochromator (Figure S1). Even though the monochromator enabled analysis at their reported excitation and emission maxima, HBC497 and HBC508 showed less fluorescence activation (1.2 and 1.3 fold) due to higher background autofluorescence in blue-green wavelengths. In contrast, HBC599 (orange emission) showed increased fluorescence activation when analyzed using a monochromator so that suboptimal emission filters likely reduced its signal in flow cytometry.

For the remaining dyes, fluorescence over no dye controls ranged from 17- to 269-fold in E. coli, 2.8–42-fold in B. subtilis, and 1.5–81-fold in S. Typhimurium (Table S1). The green emitting dyes HBC514, HBC525, and HBC530 generally gave the highest fluorescence signals. For E. coli, HBC514 was selected for further analysis, whereas for B. subtilis and S. Typhimurium, HBC530 was selected due its lower fluorescent background compared to both no dye and uninduced cell controls (Figures S2, S3). Fluorescence microscopy was performed with these dyes in E. coli, B. subtilis, and S. Typhimurium (Figure 2) and showed a strong fluorescence signal in live cells. The fluorescence micrographs show that cellular fluorescence stands in high contrast to the background even with no washing, meaning the free dye present in the solution gives minimal fluorescence compared to the fluorescent RNA–dye complex present in induced cells. Furthermore, few fluorescent cells are observed in the no IPTG condition from leaky background expression under the T7 expression system compared to the induced condition.

Figure 2.

Figure 2

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Microscopy analysis of the Pepper aptamer in live cells. Phase contrast and fluorescence micrographs of bacterial species E. coli, B. subtilis, and S. Typhimurium after incubation with green HBC dyes, HBC514 or HBC530. Expression of tRNA scaffolded Pepper was induced with 1 mM IPTG for E. coli and B. subtilis cells and 2 mM for S. Typhimurium cells. E. coli cells were stained with HBC514 (200 nM) and B. subtilis and S. Typhimurium cells were stained with HBC530 (200 nM). Scale bars represent 10 μm.

Nongreen dyes that exhibit significant fluorescence signals in all three bacterial species tested are HBC485 and HBC620, which correspond to blue and red emitting dyes. Although HBC485 and HBC620 give lower fluorescence compared to the green HBC dyes in flow cytometry analysis, it has been reported that these dyes demonstrate higher photostability in vivo, and HBC620 has been applied for super-resolution microscopy in mammalian cells.18 Fluorescence microscopy was performed with these two dyes in later experiments.

Comparison of HBC–Pepper to DFHBI-1T-Spinach2 in Different Bacteria

We next compared the brightness of the green dye–aptamer pairs, HBC–Pepper and DFHBI-1T–Spinach2, in E. coli and B. subtilis. We chose to compare HBC–Pepper against DFHBI-1T–Spinach2 as this is a well-studied dye–aptamer system that we and other laboratories have applied in bacteria.10,11,27−29 Interestingly, HBC514 was 4-fold brighter than DFHBI-1T in E. coli, whereas HBC530 was 1.5-fold brighter than DFHBI-1T in B. subtilis (Figure 3, left panels), even though only 200 nM of HBC dye was applied to cells compared to 50 μM of DFHBI-1T. The two dye–aptamer systems are orthogonal to each other, as little to no fluorescence was observed when the dyes are incubated with cells expressing the wrong aptamer (Figure 3, middle and right panels; Table S3). Taken together, these results demonstrate that HBC–Pepper is brighter than the DFHBI-1T–Spinach2 system and is orthogonal to the system for live-cell fluorescence imaging studies in both Gram-positive and Gram-negative bacteria.

Figure 3.

Figure 3

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Comparison of HBC–Pepper and DFHBI-1T–Spinach2 in Gram-negative and Gram-positive bacteria. (Top row) Representative histograms of E. coli cells expressing tRNA scaffolded Pepper or Spinach2 in the presence or absence of their respective dye partner (left panel). Center and right panels show representative histograms for the RNA aptamers with each dye to demonstrate orthogonality. (Bottom row) Same for B. subtilis cells. Aptamer-only controls with no dye are represented with a solid black line (tRNA Spinach2) and a dashed black line (tRNA Pepper).

Effects of Scaffolds on Pepper Aptamer Fluorescence

Various RNA scaffolds have been applied to fluorogenic aptamer systems to enhance the cellular fluorescence. One well-known example is the tRNALys3 scaffold, which is derived from the lysine tRNA in humans.4,25,30 Aptamers incorporated into the anticodon stem of this tRNA have increased folding and heterogeneous expression in live cells.4,25,30 Additionally, the F30 scaffold was engineered from the viral φ29 three-way junction RNA motif26 whose structure has been determined by X-ray crystallography.31 Initial application of this scaffold in live cells showed increased fluorescence as well as stability against RNA cleavage processes.26 The 3WJ scaffold was developed by modifying the structure of the F30 scaffold.32 So far, Pepper fluorescence has been tested in the tRNALys3 and F30 scaffolds.18

We designed chimeric constructs by simple grafting of the Pepper aptamer into the scaffolds (Figure 4A). Preliminary in vitro assays to measure Pepper aptamer folding in different scaffolds were conducted at 37 °C (Figure 4B, Table S2). As previously described,27 the percent of the aptamer that is competently folded for dye binding can be estimated by comparing the fluorescence signals between one sample in which the RNA is limiting and the other in which the dye is limiting, where the limiting concentration is high above the KD value to ensure saturated binding. Typically, the fluorescence in the RNA-limiting case will be lower than that in the dye-limiting case because the RNA will not be 100% folded in the active conformation. The in vitro percent folding results with different Pepper constructs and HBC514 were F30 < no scaffold < tRNA < 3WJ. In contrast, quantitative analysis of cellular fluorescence for the same constructs in E. coli by flow cytometry showed that no scaffold < 3WJ < F30 < tRNA. Thus, the in vitro folding efficiency does not correspond to the fluorescence observed in vivo, so computational prediction of the secondary structures was not pursued.

Figure 4.

Figure 4

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Analysis of the Pepper RNA aptamer in different RNA scaffolds. (A) Design of Pepper (blue) in different RNA scaffolds (black). (B) In vitro percent folding analysis of the Pepper aptamer in different RNA scaffolds. (C) Flow cytometry analysis of E. coli cells expressing the Pepper aptamer in different RNA scaffolds with HBC514 (200 nM). MFI values were determined by analyzing 30 000 cells per sample after 1 h of incubation with HBC514. Error bars represent the standard deviation of three (B) independent or (C) biological replicates. (D) Fluorescence micrographs of E. coli cells expressing tRNA-DF30 scaffolded or tRNA scaffolded Pepper after incubation with blue, green, or red HBC dyes (200 nM). Scale bars represent 10 μm.

Intriguingly, the 3WJ scaffold was found to be 192.5 ± 3% folded at 37 °C. This experiment was repeated three separate times and always yielded percent folding values of around 200%. We reasoned that this result likely is due to HBC514 binding to the 3WJ scaffold in another site than the Pepper aptamer binding pocket because a 1:1 binding stoichiometry theoretically can only give rise to a maximal fluorescence of 100%.

The flow cytometry experiments revealed that the tRNA scaffold greatly enhanced Pepper fluorescence over no scaffold (111×), whereas 3WJ and F30 scaffolds gave much more modest results (6× and 11×, respectively; Figure 4C). One benefit of the F30 scaffold is the ability to append two aptamer copies; as expected, the dimeric F30 construct (DF30) was twice as fluorescent as the same scaffold with a single Pepper aptamer (24×). Extending this rationale, we grafted F30 onto the tRNA scaffold, which retained high fluorescence in the monomeric construct (109×), while the dimeric construct was 199× brighter than the Pepper aptamer alone. The enhanced fluorescence signal also was clearly observed in fluorescence microscopy comparing the tRNA and DF30–tRNA scaffolds (Figure 4D). Furthermore, both RNA scaffolds are compatible with HBC485, HBC514, and HBC620 dyes, which are spectrally resolved from each other.

An Improved Scaffold for Dual Aptamer Display

The tRNA–DF30 scaffold has the potential to display two different aptamers, as has been shown previously for the F30 scaffold33−35 (Figure 5, Figure S4). The two aptamers HBC620–Pepper and DFHBI-1T–Spinach2 represent orthogonal dye–aptamer pairs that should be spectrally resolvable. Initial results with one copy of Pepper or Spinach2 within the F30 scaffold showed an orthogonal fluorescence signal as expected, with minimal to no crosstalk when incubated with noncognate dyes, as the background fluorescence for DFHBI-1T and HBC620 was only 1.2-fold and 1.3-fold over no dye controls, respectively (Table S4). Interestingly, when both aptamers were appended to either the DF30 or tRNA-DF30 scaffold, we observed not only that the tRNA-DF30 scaffold gave higher fluorescence, as expected, but the placement order of the aptamers on the two arms also affected the overall fluorescence output. Appending Pepper to the 5′ arm and Spinach2 to the 3′ arm of tRNA–DF30 resulted in 1.5-fold and 1.8-fold higher fluorescence for the respective aptamers compared to the opposite order. Overall, these experiments show that tRNA–DF30 is an improved scaffold for displaying two fluorogenic aptamers, including orthogonal pairs, with up to 138- and 118-fold increases in fluorescence over no dye controls.

Figure 5.

Figure 5

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Analysis of dual color constructs containing fluorogenic RNA aptamers, Spinach2 and Pepper. Flow cytometry analysis of dual color aptamer constructs containing both Spinach2 (green) and Pepper (red) and control variants in E. coli after incubation with DFHBI-1T (50 μM) and HBC620 (200 nM). MFI values from the FITC emission filter represent DFHBI-1T–Spinach2 fluorescence output, and the TxRed emission filter represents HBC620–Pepper fluorescence output and were determined by analyzing 30 000 cells per sample after a 1 h incubation with or without dyes, DFHBI-1T and HBC620. Error bars represent the standard deviation of the three biological replicates.

Discussion

In this study, we evaluated the recently developed dye–RNA aptamer pair, HBC–Pepper, for its application in live cell imaging in different bacteria. We quantitatively analyzed all HBC dye analogs in three different bacterial species through flow cytometry, which showed that different dyes were optimal for E. coli versus B. subtilis and S. Typhimurium. These results also show that the HBC–Pepper system can work in both Gram-positive and Gram-negative bacterial species. In fact, the same plasmid encoding tRNA–Pepper was applied in both E. coli and S. Typhimurium.

The HBC dyes were characterized in vitro to have similar binding affinities to the Pepper aptamer and quantum yields, but clearly our results reveal that there are dramatic differences in performance in live bacteria.18 The differences in cellular fluorescence for different dyes can be attributed to multiple factors, mainly the cell permeability of the dyes, which will vary across different bacterial species. Also, the flow cytometer used in the dye screen has a limited set of excitation lasers and emission filters, which reduced the observed fluorescence for HBC599, whereas HBC 497 and HBC508 still showed minimal fluorescence when they were measured using a monochromator. However, the main takeaway is that HBC dyes in different color ranges give useful cellular fluorescence signals in E. coli, B. subtilis, and S. Typhimurium, including green dyes HBC514 and HBC530 and the more photostable nongreen dyes HBC485 and HBC620.

Similar to what we previously observed for the Spinach2 aptamer,36 the tRNA scaffold greatly enhances cellular fluorescence of the Pepper aptamer. Our results show that it is a superior scaffold for bacterial cells compared with others that have been previously utilized primarily in mammalian cells. We also found that cellular fluorescence does not correlate with in vitro folding efficiency for the Pepper aptamer in different scaffolds. The magnitude of the observed effects (e.g., 110× difference between tRNA versus no scaffold) suggests that it is most likely due to differences in cellular expression and/or stability. Unlike the other tested scaffolds, natural tRNAs contain internal promoter sequences and are end processed, chemically modified, and bound by proteins; each of these steps can lead to enhanced expression, stability, and/or folding efficiency of tRNA-scaffolded constructs but are difficult to recapitulate in vitro.

Finally, the development of the DF30–tRNA scaffold results in an optimal construct that is 199 times brighter than the Pepper aptamer alone in E. coli cells. Researchers previously have made linear arrays of fluorogenic aptamers to enhance the fluorescence signal toward single-molecule imaging of RNAs,13,19,37,38 but they are not directly additive for signal enhancement because of misfolding. Our results highlight that novel scaffolds such as DF30–tRNA are an important strategy to pursue because they provide much greater than additive enhancement of fluorescence signal and can be applied readily to different fluorogenic aptamers. Extending the rationale of DF30–tRNA even further to a “dendrimer”-like design may lead to better separate folding of each aptamer and thus require fewer aptamer copies for single-molecule imaging.

Ongoing work seeks to determine whether the 5′ and 3′ ends of the DF30–tRNA scaffold are processed and cleaved like tRNA. Very recently, researchers demonstrated Forster resonance energy transfer (FRET) between the DFHBI-1T–Broccoli (related to Spinach2) and HBC620–Pepper aptamers in vitro using a three-helix RNA origami scaffold that also enabled cryo-EM structure determination, showing the effects and advantages of designable scaffolds.22 Our study has developed the DF30–tRNA scaffold, which we envision can be utilized for diverse synthetic biology applications, including promoter studies and the development of scaffolded RNAs that undergo FRET, ratiometric sensors, and multiplex biosensing in bacterial cells.

Acknowledgments

This work was supported by grants from the National Science Foundation (1815508 to M.C.H.) and the National Institutes of Health (GM124589 to M.C.H. and GM056141 to K.H.). The flow cytometry core facility is supported by the National Institutes of Health award S10OD026959.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00009.

  • Additional figures and experimental methods (PDF)

Author Contributions

M.M.M. and F.C. conducted the research. M.M.M., F.C., and M.C.H. designed the experiments. M.M.M. and M.C.H. wrote the manuscript, and K.H. and M.C.H. supervised the project. All authors reviewed and edited the manuscript.

The authors declare no competing financial interest.

Supplementary Material

sb4c00009_si_001.pdf (596.7KB, pdf)

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