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. 2024 Aug 5;19(8):1836–1841. doi: 10.1021/acschembio.4c00398

Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging

Kfir B Steinbuch 1, Deyuan Cong 1, Anthony J Rodriguez 1, Yitzhak Tor 1,*
PMCID: PMC11334113  PMID: 39101365

Abstract

graphic file with name cb4c00398_0007.jpg

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3thG, result in highly emissive 5′-labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentration-gradient conditions, which was suppressed by the addition of guanosine.

The development of fluorescent nucleoside analogs is frequently challenged by their intended applications.1,2 Modifying the heterocyclic skeleton of the practically dark canonical purines and pyrimidines to endow them with useful photophysical features frequently renders them too perturbing.3−5 Even surgical structural changes, such as a single atom replacement, intended to alter their photophysics, perturb both the ground and excited states, their solvation and dynamics, and their susceptibility to distinct quenching pathways, frequently in an unpredictable fashion.6 Advancing such useful biophysical tools has therefore been an empirical and iterative process.1,7

While minimal alterations of the native heterocycles have yielded diverse families of useful analogs, including two emissive RNA alphabets reported by us (Figure 1), efforts are still required to expand their repertoire and application landscape.8−11 We have recently assessed the incorporation of hydrophobic groups into the purine skeleton at the position equivalent to the purines’ N7 and have hypothesized that desolvation of the Hoogsteen face may impact nonradiative decay pathways and lead to improved photophysical parameters (Figure 1).12 We set out to test this by modifying thG, a useful and thoroughly studied fluorescent G surrogate,10,13 by incorporating a trifluoromethyl group into its skeleton (Figure 1). Here, we report its synthesis, exploiting Baran’s heteroaromatic trifluoromethylation protocol,14,15 and evaluate its photophysical features, as well as its cellular compatibility and potential for cellular imaging.

Figure 1.

Figure 1

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Guanosine and its previously reported emissive analogs, thG, tzG, mthG, and the current CF3thG.

Results and Discussion

Instead of bottom-up synthesizing the intended target nucleoside, we have opted to exploit Baran’s trifluoromethylation procedures and directly modify the emissive thG.14,15 Since rather few examples of trifluoromethylating purine-like heterocycles and nucleosides have been reported, conditions were therefore first screened, including the use of Zn vs Na trifluoromethanesulfinate salts, as well as solvent combinations and reaction temperatures (Scheme 1, SI section 1).16 Yields were relatively low across the board (10–18%) but were deemed acceptable for the purpose of assessing the fundamental photophysics and utility of this new nucleoside.

Scheme 1. Synthesis of CF3thG.

Scheme 1

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Reagents and conditions. (a) NaSO2CF3 1.5 equiv, tBuOOH 1 equiv, DMSO, 18.5%. (b) Ac2O, DMAP, Py, 90.0%. (c) (i) NaSO2CF3 or Zn(SO2CF3)2 3 equiv, tBuOOH 5 equiv, DMSO, 15.5%. (ii) NH4OH/MeNH2 1:1, quantitative. Bottom right: crystal structure of CF3thG.

X-ray crystal structure analysis of the emissive analog confirmed the β anomeric configuration and demonstrated that in the solid state, CF3thG displays anti orientation, as native guanosine does.17 Overlay of the crystal structure of CF3thG with that of guanosine shows negligible impact of the modified nucleosbase on the sugar pucker (rmsd of 0.009 Å) and high overall similarity to the native nucleoside (rmsd 0.101 Å), including the same tautomeric preference (Figure S1).17

The absorption and emission spectra of the analytically and structurally characterized CF3thG, as well as their sensitivity to changes in polarity and pH, were recorded (Figure 2, Table 1). Both the absorption and emission maxima of CF3thG in water (343 and 475 nm, respectively) were red-shifted compared to thG (Figure 2a and Table 1).10 Unlike the complex spectra displayed by thG, due to its ground-state tautomerization,18CF3thG displays simpler spectra suggesting one predominant tautomer. The emission spectrum of thG displays a maximum and a shoulder that peak at 453 and 400 nm, respectively, when excited at its absorption maximum at 321 nm (Figure S2).10 This is due to the presence of two ground-state tautomers H-1 and H-3, which could also be selectively excited (Figure S2).18 The emission spectrum of CF3thG is simpler and displays only a small shoulder below 400 nm, suggesting a minimal contribution from a minor tautomer. To test this hypothesis, emission spectra excited at higher and lower energies were recorded. Excitation at higher energy (310 nm) resulted in a second emission peak with a maximum of around 384 nm, while excitation at lower energy (390 nm) resulted in a spectrum that aligns with the emission spectrum of CF3thG when excited at its absorption maximum (343 nm) (Figure S2). This suggests the presence of two ground-state tautomers in solution, with the H-1 CF3thG being the dominant one, as inferred from the observations made for thG.18

Figure 2.

Figure 2

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(a) Absorption (dashed lines) and emission (solid lines) spectra of tzG (orange), thG (red), mthG (gray), and CF3thG (blue) in water; inset: expansion of Abs/em around 400 nm demonstrating CF3thG still absorbs above 400 nm, which could facilitate its visualization in confocal microscopy using 405 nm excitation; (b) absorption maxima variation of CF3thG versus pH, inset: absorption spectra of CF3thG at pH 1 (black) to 13 (light blue); (c) absorption (dashed lines) and emission (solid lines) spectra of CF3thG in water (blue), dioxane (light blue), and mixture thereof (80, 60, 40, and 20% v/v water in dioxane); (d) Stokes shift correlation versus solvent polarity (ET(30)) of water/dioxane mixtures for CF3thG with standard errors. Note: the data point at 35.3 kcal mol–1 (100% dioxane) was excluded from the linear fit. Emission intensities were corrected to reflect an optical density of 0.1 at λmax. All photophysical measurements were performed in at least three replicates.

Table 1. Photophysical Data for Emissive Guanosine Analogs.

  solvent λabs [ε]a λem [Φ]a Φε Stokes shifta polarity sensitivityb pKac
tzG water 333 (4.9) 459 (0.25) 1203 8.27 102.0 3.55, 8.51
  dioxane 339 (4.7) 425 (0.17) 539 6.01    
thG water 321 (4.1) 453 (0.46) 1909 9.58 107.2 4.4, 10.3
  dioxane 333 (4.5) 424 (0.50) 2265 6.89    
mthG water 327 (3.5) 456 (0.42) 1470 8.70 130 5.0, 10.4
  dioxane 334 (3.6) 443 (0.61) 2200 7.20    
CF3thG water 343 (2.9) 475 (0.54) 1566 8.10 89 3.66, 9.44
  dioxane 354 (3.3) 450 (0.61) 2013 6.02    
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a

λabs, λem, ε, and Stokes shifts are reported in nm, nm, 103 M–1 cm–1, and cm–1, respectively. All photophysical values reflect the average of at least three independent measurements.

b

Sensitivity to polarity, expressed in cm–1/(kcal mol–1), is equal to the slope of the linearization depicted in Figure 2d.

c

pKa values are equal to the inflection points determined by the fitting curve in Figure 2b. Values for tzG, thG, and mthG were taken from refs (10)–11, respectively. See Table S3 for data including experimental errors for CF3thG.

While its extinction coefficient is lower, the higher emission quantum yield of CF3thG (ϕ = 0.54) results in comparable brightness to the parent nucleoside thG as well as to tzG and mthG (Table 1).10−12 Compared to spectra taken in water, batho- and hyperchromic shifts were observed for absorption spectra of CF3thG in dioxane, and hypso- and hyperchromic shifts were observed for the emission maxima (Table 1), associated with a slightly higher emission quantum yield (ϕ = 0.61). To determine its ground-state pKa values, absorption spectra of CF3thG were recorded in buffered solutions at pH 1–13 (Figure 2b). By fitting a double Boltzmann sigmoidal curve to the plot of absorption maxima vs pH, two pKa values of 3.7 and 9.4 were extracted. As expected, these values are lower than those of thG and mthG and are closer to the more isomorphic analog tzG due to the electron-withdrawing inductive effect of the trifluoromethyl group.10−12

Sensitivity to microenvironmental polarity was determined by measuring the absorption and emission of CF3thG in water/dioxane mixtures (Figure 2c). Correlating the observed Stokes shifts vs Reichardt’s ET(30) parameters (Figure 2d) shows comparable behavior to our other emissive guanosine analogs (Table 1).19

To study the effect the CF3 moiety has on the photophysics of CF3thG, spectra were taken in trifluoroethanol (TFE) and compared to other alcohols and water. Stokes shifts of CF3thG in the different alcohols and water were plotted against their ET(30) values20 and against their hydrogen-bond strength α1 parameter21 (Figure S3, Table S5). The linear correlations observed show that no solvent-specific effects impact the photophysics of CF3thG (Figure S3b,c, Table S5). To further probe this matter, the absorption and emission spectra of CF3thG in H2O vs D2O were compared22 and were found nearly identical (Figure S4). These results, therefore, further suggest that the inductive electronic effect of the CF3 moiety is dominating the photophysics of CF3thG over its environmental- and solvent-specific effect.

The ability of CF3thG, as a free nucleoside, to initiate in vitro transcription reactions using T7 RNA polymerase was evaluated and compared to GTP (Figure 3).23−25 Using the nucleoside and not the nucleotide facilitates the generation of strands labeled only at the 5′ position.26 Transcripts obtained were separated by PAGE and visualized under UV light. For the in vitro transcription reaction containing CF3thG, bands corresponding to GTP initiation (T1) and CF3thG initiation (T2) were visible by UV shadowing. The latter band was also fluorescent and hence visible upon illumination at 365 nm (Figures 3 and S5).27,28 The extracted transcripts were characterized by their emission spectra and MALDI-TOF MS analyses, supporting their assigned sequences (Figure 3). The desired, highly emissive CF3thG-initiated strand was quantified by its absorption at 260 nm and was found to be in ∼8-fold excess of the corresponding template DNA duplex. Its relative yield, compared to that of the native transcript formed in the same transcription reaction, was 0.38.

Figure 3.

Figure 3

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Transcription reactions with the T7 RNA polymerase. (a) T7 promoter and template strand sequences; (b) transcription reaction using the template with 2 mM of all-natural NTPs, with or without CF3thG (10 mM). The white arrows indicate the target native transcript, T1, and the CF3thG-initiated transcripts, T2. UV shadowing and photoluminescence (PL) were observed upon illumination at 254 and 365 nm, respectively; (c) absorption (dashed line) and emission (solid line) of T2; (d) MALDI-TOF mass spectrum of transcript T2.

To probe the imaging potential of CF3thG, its cytotoxicity and cellular uptake were first evaluated. MTT cell viability assay demonstrated that CF3thG, as well as the other analogs, mthG, thG, tzG, and the native guanosine, did not have a detrimental effect on the cell viability of HEK293T cells up to the highest tested concentration (500 μM) after 24 h incubation (Figure S6).29

Following calibration and optimization experiment using widefield microscopy (Figures S8 and S9), confocal imaging illustrated that CF3thG could be clearly visualized in live HEK293T cells using a 405 nm laser excitation, while mthG, thG, and tzG could not be detected (Figures 4a and S10). CF3thG was observed in the nucleus and the cytosol. In the nucleus, it appeared to localize into spherical structures, perhaps the nucleolus, where the synthesis of ribosomes takes place.30 In the cytosol, most of the signal by CF3thG adopted a distinct pattern spreading throughout the cell rather than a uniformly diffused signal. To determine if CF3thG colocalizes with certain cellular organelles, cells incubated with CF3thG were also stained with lyso-tracker, Mito-tracker, ER-tracker, and lipid droplet markers (Figure S11). Results show that the pattern of CF3thG moderately colocalizes with the ER, with a Pearson colocalization factor of 0.35 (Figure 4b, SI section 5.4).31 The Mander’s colocalization coefficients indicate that the signal of CF3thG is found in the ER and the lysosomes, as their fractions of signals overlapping with the signal of CF3thG are 0.999 and 0.98, respectively (Figure 4b, SI section 5.4).31

Figure 4.

Figure 4

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Live cell imaging of HEK293T incubated with (a) tzG, thG, mthG, or CF3thG (500 μM) for 24 h (merged channels); (b) CF3thG (500 μM) for 24 h and stained with an ER-tracker for 30 min. To visualize the cells, nuclei were stained with NucRed for 30 min. Figure 4 represents the results of at least two independent experiments.

Finally, real-time live cell imaging of CF3thG over the course of 30 min captured the movements of CF3thG in the cells as well as its expulsion (Figure 5a, Sup Movie 1). It was hypothesized that CF3thG could be expelled from the cells due to concentration gradients as the samples were washed, and an imaging medium was applied prior to microscopic visualization. An identical experiment in which native guanosine (500 μM) was added to the imaging media supported this hypothesis, as cellular CF3thG was better retained over 30 min (Figure 5b, Sup Movie 2). Exosomal release of guanosine by muscle satellite cells, which could be delivered to the central nervous system has been previously reported.32,33 While additional experimentation is necessary, it is plausible that CF3thG is capable of visualizing endogenous phenomena associated with the release of guanosine derivatives via exosomes.

Figure 5.

Figure 5

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Real-time live cell imaging of HEK293T cells incubated with CF3thG (500 μM) for 24 h. After incubation, cells were washed twice and visualized in Fluorobrite (a) or Fluorobrite supplemented with guanosine (500 μM) (b) for 30 min. Nuclei were stained with NucRed. The experiment’s results were reproduced in two independent experiments.

In summary, we introduce CF3thG, a new emissive guanosine analog, synthesized by direct trifluoromethylation of thG or ActhG based on Baran’s trifluoromethylation methods. T7 RNA polymerase was found to initiate in vitro transcription with CF3thG, resulting in highly emissive 5′-end-tagged RNA transcripts, suggesting that the new emissive analog, despite its large Hoogsteen face substituent, can still be recognized by polymerases. While other emissive nucleosides have been recently reported to facilitate live cell imaging, it appears that these analogs could be visualized only when embedded into RNA strands.34−36 Here, we demonstrate that due to its intense visible emission, CF3thG can be visualized as the free nucleoside in live mammalian cells and could be monitored in real time. Imaging experiments with and without guanosine allude to its similarity to guanosine. Further studies, however, are needed to support that the physicochemical differences between CF3thG and G do not significantly alter the nucleoside’s cellular biochemistry, localization, and metabolism. Having a guanosine-like probe that can be visualized while being taken and released from the cells, as shown here, could potentially assist in shedding light on cellular processes involving guanosine.37−40

Methods

General Chemistry Methods

Reagents, buffers, and salts were purchased from Sigma-Aldrich, Fluka, TCI, Acros, Synchem, Inc. (Elk Grove, IL), and were used without further purification unless otherwise specified. NTPs were purchased from Fisher. Template and promoter oligonucleotides were purchased from IDT. T7 RNA polymerase (P266L) was expressed and purified as previously reported.25

Solvents were purchased from Sigma-Aldrich and Fisher Scientific and dried by standard techniques. NMR solvents were purchased from Cambridge Isotope Laboratories (Andover, MA). All reactions were monitored with analytical TLC (Merck Kieselgel 60 F254). All experiments involving air and/or moisture-sensitive compounds were carried out under an argon atmosphere. Column chromatography was carried out with a silica gel particle size of 40–63 μm. NMR spectra were obtained on Bruker AVA 300 MHz, Varian Mercury 400 MHz, and Varian VX 500 MHz. MALDI-TOF mass spectra were obtained on a Bruker Autoflex Max MALDI-TOF-MS. ESI-TOF mass spectra were obtained on an Agilent 6230 HR-ESI-TOF MS instrument at the Molecular Mass Spectrometry Facility at the UCSD Chemistry and Biochemistry Department.

General Photophysics Methods

Spectroscopic grade DMSO and dioxane were obtained from Sigma-Aldrich, and aqueous solutions were prepared with Milli-Q deionized water. All measurements were carried out in a 1 cm, four-sided quartz cuvette from Helma. All measurements were reproduced in at least 3 independent experiments.

Absorption spectra were measured on a Shimadzu UV-2450 spectrophotometer, setting the slit at 1 nm and using a resolution of 0.5 nm. All spectra were corrected for the blank. Steady-state emission spectra were measured on a Horiba Fluoromax-4 instrument setting the excitation and the emission slits at 1 and 3 nm, respectively, and the resolution at 1 nm. Both instruments were equipped with a thermostat-controlled ethylene glycol water bath fitted to a specially designed cuvette holder, and the temperature was kept at 25.00 ± 0.10 °C. All spectra were corrected for the blank.

Nucleosides (tzG, thG, mthG, and CF3thG) were dissolved in DMSO to prepare highly concentrated stock solutions (10 mM). In a typical experiment, aliquots were diluted into solvents to a final volume of either 125 μL or 3 mL.

Cell Biology and Confocal Microscopy

HEK293T cells were grown in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. Cells were grown in a humidified chamber with 95% air and 5% CO2 at 37 °C. 96-well microtiter plates and Fluorodish wells were coated with PDL before use according to the following procedure: Wells were covered with PDL solution (0.1 mg mL–1, 100 or 120 μL per well for 96-well plate or fluorodish well, respectively) and incubated for 3 h at RT. The PDL solution was then removed by aspiration, and wells were resterilized under UV light for 20 min and further dried for an additional 1.5 h. Wells were finally washed twice with PBS (100 or 120 μL per well).

Live cell imaging was performed using a Nikon AXR confocal microscope with four lines (405, 488, 561, and 640 nm) LUA-S4 laser engine and DUX-VB detector using bandpass and long-pass filter for each channel (420–551, 655–850 nm) mounted on a Nikon Ti2 using an Apo 100 × 1.45 NA objective and operated using NIS elements 5.42.03 software. Image stacks were acquired in Galvano mode in unidirectional scanning with a 405 nm laser at 3% power and 61.5 μm pinhole size and 640 nm laser at 2% power and 61.0 μm pinhole size at a frame size of 1024 × 1024 at scan zoom 1.7. Cells were maintained at 37 °C and 5% CO2 with 80% humidity using an okolab bold line.

Acknowledgments

We thank the National Institutes of Health for their generous support (GM139407), the Chemistry and Biochemistry MS Facility, and the UCSD X-ray crystallography Facility. Confocal microscopy was performed at the Nikon Imaging Center at UC San Diego. We would like to thank R. Sanchez and P. Guo for their support.

Glossary

Abbreviations

PAGE

poly(acrylamide gel electrophoresis)

MTT

3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide

ER

endoplasmic reticulum

ActhG

per-acetylated thG

TFE

trifluoroethanol

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.4c00398.

  • Synthetic procedures; photophysical data; in vitro transcription; cell biology; and fluorescence microscopy protocols; movies of real-time imaging timelapse (PDF)

  • Sup Movie 1 (ZIP)

  • Sup Movie 2 (ZIP)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

National Institutes of Health (GM139407).

The authors declare no competing financial interest.

Supplementary Material

cb4c00398_si_001.pdf (1.3MB, pdf)
cb4c00398_si_002.zip (97.5MB, zip)
cb4c00398_si_003.zip (122.9MB, zip)

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Supplementary Materials

cb4c00398_si_001.pdf (1.3MB, pdf)
cb4c00398_si_002.zip (97.5MB, zip)
cb4c00398_si_003.zip (122.9MB, zip)

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