Brightly Fluorescent 2â²-Deoxyribonucleoside Triphosphates Bearing

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Brightly Fluorescent 2'-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in cellulo Incorporation to DNA, Imaging and Flow Cytometry Pedro Güixens Gallardo, Zbigniew Zawada, Jan Matyasovsky, Dmytro Dziuba, Radek Pohl, Tomas Kraus, and Michal Hocek Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00721 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 28, 2018

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Bioconjugate Chemistry

Brightly Fluorescent 2'-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in cellulo Incorporation to DNA, Imaging and Flow Cytometry Pedro Güixens Gallardo,†,‡ Zbigniew Zawada,† Ján Matyašovský,†,‡ Dmytro Dziuba,† Radek Pohl,† Tomáš Kraus,† and Michal Hocek†,‡* †

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo

nam. 2, CZ-16610 Prague 6, Czech Republic. ‡

Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8,

CZ-12843 Prague 2, Czech Republic.

Abstract: Synthesis of cytosine, uracil and 7-deazaadenine 2'-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenylbodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labelled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dNmBdpTPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labelled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dNmBdpTPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dCmBdpTP was used to monitor mitosis in live cells. TOC graphic:

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Introduction Fluorescent labelling of nucleotides and nucleic acids is an indispensable tool in chemical and molecular biology.1-3 Environment-sensitive fluorophores are used as probes for changes of secondary structure or for sensing biomolecular interactions.4-10 On the other hand, labelling of nucleic acids with bright and photostable fluorophores is needed for applications in imaging, microscopy and metabolic studies11-15 as well as for sequencing.16 Many diverse fluorophorelabelled 2'-deoxyribonucleoside triphosphates (dNTPs) were reported in literature.17-23 Most of them bear cyanines, fluoresceins and other bulky charged labels with limited photostability linked through a flexible tether at position 5 of dUTP, and some are even commercially available. They are used for enzymatic labelling of DNA through polymerase incorporation of modified nucleotides.12,20,23-26 Since some of these bulky modified dNTPs are rather poor substrates for DNA polymerases and inherently they do not penetrate cell membranes, they cannot be directly used for in vivo or in cellulo incorporation to DNA, except for some studies involving highly invasive techniques temporarily disrupting the integrity of plasma membrane or fixed cells.27-31 Very recently, Zawada et al. reported32 the first synthetic nucleoside triphosphate transporter SNTT (Figure 1) which enabled efficient and rapid intracellular delivery of modified dNTPs and their incorporation into genomic DNA for direct fluorescent staining and imaging using some commercially available fluorescent dNTPs. However, some commercial and widely used dNTPs with bulky fluorophores, e.g. AF488-dUTP, were found32 to be poorly incorporated into DNA in cellulo. Therefore, there was a need to develop new brightly fluorescent dNTPs bearing reasonably small fluorophores to ensure their efficient incorporation by the endogenous DNA replication machinery. Here we report on new methylated bodipy-linked dNTPs and their use in in cellulo labelling of the genomic DNA for applications in fluorescence microscopy and flow cytometry. Fluorophores based on difluoro-boraindacene core (4,4-difluoro-4-borata-3a-azonia-4a-aza-sindacene, bodipy) are attractive and useful dyes with good photostability and high brightness.3335

Bodipy fluorophores were widely used for the labeling of biomolecules including nucleic acids.

Fluorescent deoxyuridine analogues based on the bodipy fluorophore and their incorporation into DNA via solid-phase phosphoramidite synthesis were reported.36–38 Commercial dUTP 2 ACS Paragon Plus Environment

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derivatives with BODIPY fluorophore tethered via long flexible linker were used for in cellulo fluorescence labeling of DNA and for detection of apoptotic cells. 27-29 We have recently reported8,39 dCTP and 7-deaza-dATP derivatives bearing meso-phenyl bodipy and their use in polymerase synthesis of modified DNA. The phenyl-bodipy served as a molecular rotor with excellent sensitivity to viscosity and the labelled oligonucleotides (ONs) and DNA were used as fluorescence life-time sensors for interaction with lipids or proteins.8 However, for cellular imaging applications, we needed brighter fluorophores insensitive to any environment changes. Therefore, we have chosen hexamethylated phenyl-bodipy which is known40 to have high quantum yields in any environment due to restricted rotation.41

Results and Discussion Synthesis The synthesis of target hexamethylbodipy-linked nucleosides and dNTPs was envisaged through the Sonogashira cross-coupling22,25 of the corresponding propargyl-linked hexamethylbodipy with halogenated nucleosides (Scheme 1). The propargylated fluorophore 2 was synthesized by alkylation of known42 bodipy-phenol 1 with propargyl bromide in presence of potassium carbonate in analogy to literature procedures.43,44 The Sonogashira coupling of alkyne 2 with 5iodo-2'-deoxyuridine (dUI), -cytidine (dCI) or 7-iodo-7-deaza-2'-deoxyadenosine (dAI) was performed in presence of CuI, triethylamine and Pd(PPh3)2Cl2 in DMF to afford the corresponding fluorophore-linked nucleosides dUmBdp, dCmBdp and dAmBdp in good to excellent yields of 65, 97 and 91%, respectively. The triphosphorylation of nucleosides dNmBdps was performed under standard conditions45 by treatment

with POCl3, followed by pyrophosphate

and

triethylammonium bicarbonate (TEAB) to furnish the target labelled triphosphates dUmBdpTP, dCmBdpTP and dAmBdpTP in 20, 40 and 24% yields, respectively, after purification by HPLC and ion exchange.



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Scheme 1. Synthesis of labelled nucleosides and dNTPs

Spectroscopic and photophysical properties of labelled nucleosides and dNTPs The UV-vis absorption and fluorescence emission properties of the labelled nucleosides dUmBdp, dCmBdp and dAmBdp and triphosphates dUmBdpTP, dCmBdpTP and dAmBdpTP are summarized in Table 1 (also see Figures S1-S3 in SI). All compounds display strong absorbance in the visible region with a band centered at 498-500 nm and all of them show bright fluorescence with emission maxima at 507-508 nm. The fluorescence quantum yields of all modified nucleosides in ethanol were very high (95-98%). The quantum yields of the corresponding dNmBdpTPs in water differed quite significantly. While the 7-deazaadenine derivative dAmBdpTP gave a moderate quantum yield of 45%, the modified uracil dUmBdpTP and (even more) cytosine dCmBdpTP nucleotides displayed high 4 ACS Paragon Plus Environment

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to very high quantum yields of 86 and 92%, respectively. The nucleosides and nucleotides displayed excellent overall brightness up to 70,000 M-1cm-1. This indicates, that these fluorophore-linked nucleotides show promising potential for bright fluorescent labelling of DNA in biological environment. Table 1. Spectroscopic and photophysical properties of all new fluorescent compounds Compound

Solvent

2

EtOH

dAmBdp

dCmBdp

dUmBdp

dAmBdpTP

dCmBdpTP

dUmBdpTP

a

EtOH

EtOH

EtOH

buffer(f)

buffer

buffer

λabs

ε

λem

Φf (d)

[nm] (a) [104 M-1 cm-1] (b) [nm] (c) 500

10.3

306

0.6

500

6.8

280

1.4

500

7.3

303

1.1

500

5.2

292

1.0

498

7.5

279

1.9

498

7.4

298

1.6

498

7.1

296

1.2

B [104 M-1 cm-1] (e)

508

0.97±0.02

10.0

508

0.98±0.03

6.7

508

0.96±0.01

7.0

508

0.95±0.01

4.9

507

0.45±0.03

3.4

507

0.92±0.02

6.8

507

0.86±0.02

6.1

absorption maximum (±1 nm); b confidence interval did not exceed ±0.2 104 M-1 cm-1; c emission maximum

(±1 nm); d quantum yield of fluorescence measured using fluorescein in 0.1 M NaOH (Φf = 0.92 at 25 °C) as a standard; e B = ε × Φf; f phosphate buffer (20 mM, pH = 7.0, 1 M NaCl)

Polymerase incorporation of labelled nucleotides The modified dNmBdpTPs were tested as substrates for DNA polymerases in primer extension using a 15-nt primer and 31-nt template with 5'-TINA cap to inhibit non-templated incorporation46 (PEX, Figure 1; for sequences of all ONs, see Table S1 in SI). The KOD XL DNA 5 ACS Paragon Plus Environment


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polymerase was found to efficiently incorporate the modified nucleotides into the extended primer to form full-length 31-mer dsDNA product containing four modifications (Figure 2). Other B-family DNA polymerases, i.e. KOD (exo-) and Vent (exo-), as well as A-family polymerase Bst Large fragment, could also incorporate the modified nucleotides (see Figures S4-S6 in SI). In some of these cases, the TINA-capping of template reduced but not fully eliminated the non templated addition and we also observed some n+1 products. The identity of all extended ONs was confirmed by MALDI of ssON formed by PEX with biotinylated template followed by magnetoseparation (Table 2).47 Although the mobility of ON4AmBdp (product of PEX with dAmBdpTP) seems to be slightly faster than the other PEX products, the MALDI confirmed the correct length and sequence of this labelled ON as well. We also performed a simplified kinetic study of single nucleotide extension using modified dNmBdpTPs in comparison with the natural dNTPs (Figure S7 in SI) and found that the extension of primer with the modified dNmBdp nucleotides is somewhat slower (2 min. for dCmBdp and dUmBdp or 5 min. for dAmBdp) than extension with the natural nucleotides (completed within 0.5-1 min.).


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Figure 1. (a) PEX with modified dNmBdpTPs; (b) transport of dNmBdpTPs into cells and labelling of genomic DNA; (c) structure of SNTT transporter.



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Figure 2. Primer extension experiment in the presence of KOD XL DNA polymerase using tempPrb4basII-TINA. Lane 1 and 9, P: primer; lane 2, +: product of PEX with natural dNTPs; lane 3, A: products of PEX with dTTP, dCTP and dGTP; lane 4, C-: products of PEX with dTTP, dATP and dGTP; lane 5, U-: products of PEX with dATP, dCTP and dGTP; lanes 6-8, Nmbdp: product of PEX with functionalized dNmbdpTP and corresponding three natural dNTPs.

Table 2. MALDI data of modified ONs ON

M (calcd) / Da

M (found) [M+H] / Da

ON4AmBdp

11230.5

11229.6

ON4CmBdp

11234.5

11232.9

ON4UmBdp

11178.5

11177.7

Then we tested the modified dNmBdpTPs in PCR amplificantion using a 98-mer template. We found that the PCR with any of these dNmBdpTPs completely replacing the corresponding natural dNTP did not work. The difficulty is probably not in the incorporation of the modified nucleotides, but in efficient reading of the polymerase through hypermodified template, although a literature example48 reports that DNA polymerase can extend primer when template is modified by bodipy. However, we tested different ratios of dNmBdpTP/dNTP and found that 8 ACS Paragon Plus Environment

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when using the modified dNmBdpTPs in presence of the corresponding natural dNTP in ratio 1:1, the PCR amplifications were efficient and the resulting modified DNA was brightly fluorescent (Figures S11-S22 in SI). PCR amplification using an even more challenging longer 331-mer template containing several oligo-A, oligo-C and oligo-T stretches was also tested. In this case, the amplifications worked only with lower proportion of the modified dNmBdpTPs (5-20%) but due to the high quantum yields of the incorporated dNmBdp nucleotides the PCR products were still visualized using fluorescent scanner without the necessity of external dyes. These results indicate that these fluorescent dNmBdpTPs are reasonably good substrates for DNA polymerases and can be at least partially incorporated even in the presence of the natural dNTPs (in accord with our recent works49,50 showing that alkyne-linked dNTPs are efficient in competition primer extension in presence of natural dNTPs). Therefore, the dNmBdpTPs can be potentially incorporated into genomic DNA if they can be efficiently transported into the living cells.

Transport into cells and in cellulo incorporation In order to study the intracellular delivery of dNmBdpTPs and incorporation of modified nucleotides into genomic DNA, synchronized (24-h pre-incubation with hydroxyurea) or nonsynchronized U2-OS cells were treated with solution of transporter SNTT and either dNmBdpTP or commercial dUTP-Alexa-488 for two minutes. Cells were then grown in complete medium up to five days and analyzed either by confocal microscopy or by cytometry in indicated times after addition of the medium.



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Figure 3. Comparison of efficiency of fluorescent staining and labelling of genomic DBA using different dNmBdpTP in presence of SNTT in hydroxyurea-synchronized U2-OS cells. Confocal images for three dNmBdpTP: dCmBdpTP (A: after 1 h, B: after 5 h), dUmBdpTP (C, after 5 h), dAmBdpTP (D, after 5 h). Scale bar 20 µm. Images were thresholded to improve contrast of each picture.

In synchronized cells, the confocal microscopy showed that all three dNmBdpTP were efficiently transported to the cells within 2 minutes and the nucleotides eventually got incorporated into DNA (Figure 3), however the efficiency of the process differed significantly. The dCmBdp nucleotide was incorporated quickly and most efficiently. Even after 1 h, we observed strong nuclear foci (spotted fluorescence pattern inside the nuclei as a sign of DNA incorporation, Figure 3A), whereas in 5 h the foci were even better visible while much weaker fluorescence was left in the cytosol (Figure 3B). The dUmBdp nucleotide was incorporated less efficiently (Figure 3C), while the 10 ACS Paragon Plus Environment

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dAmBdp nucleotide was incorporated only sparingly (Figure 3D). On the other hand, the commercially available dUTP-Alexa-48851,52 was transported somewhat less efficiently (less homogeneous staining within cell population, Figure S23C), the fluorescence staining occurred in the whole cells (both in nuclei and cytosol) even after 20 h and any potentially formed nuclear foci were barely visible due to background fluorescence (Figure S24D in SI). This makes it impossible to use Alexa-488 derivative for detection of DNA synthesis in live imaging because of the strong background fluorescence of unincorporated Alexa-488. The fluorescent labelling of genomic DNA by dCmBdp did not significantly compromise the viability of the cells that can undergo mitosis, which can be monitored by confocal microscopy (Figure 4). We were able to follow up to 2-3 cell divisions while still keeping significant fluorescence.

Figure 4. Mitosis time-lapse in U2-OS cells with incorporated dCmBdp nucleotide at 25 h after treatment. Scale bar is 20 µm. Only part of mitosis is shown (starting from metaphase). Thresholded from 0-255 to 2-34. 11 ACS Paragon Plus Environment


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In the non-synchronized cells, the dCmBdpTP was also quickly transported and efficiently incorporated into DNA in the cells synthesizing DNA, whereas in the cells not synthesizing DNA, the fluorescence stayed mainly in the cytosol retaining the nuclei dark. This strong contrast (fluorescent nuclear foci with dark cytosol versus dark nuclei with fluorescent cytosol, Figures S23A,B and S25-S27 in SI) can be used for distinguishing between cells in S-phase of cell cycle from cells in other phases. Thus the staining of genomic DNA solely with dCmBdp nucleotides gives significant advantage in live cell imaging without the need of counterstaining of nuclei of even cell fixation. In non-synchronized cells, after 20 h, ca. 40% of cells showed strong fluorescence of the chromatin and dim cytosol in case of dCmBdpTP (Figure S24A and S25 in SI), whereas the use of dUmBdpTP and dAmBdpTP resulted in lower proportion of the cells with fluorescently labelled chromatin (20 % and 10%, respectively, Figures S26,S27). Note however that these numbers are just indicative since they depend significantly on medium, temperature, cell confluency, time from the seeding of the cells, etc. The apparent weakest nuclear staining with dAmBdp nucleotide should not be attributed to its lower quantum yield (Table 1) but rather to its weakest incorporation into chromatin. The differences in the efficiency of chromatin staining could be explained by a combination of several factors including different stability of the dNmBdpTP in the cell, different transport of the modified dNmBdpTP into nuclei, as well as different substrate activity to human replicative DNA polymerases. In any case, the dCmBdpTP as the best performing building block in all cell-based experiments giving always the fastest and most efficient staining of the chromatin.


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Figure 5. Evidence of incorporation of modified dNmBdp nucleotides to genomic DNA by flow cytometry in U2-OS cells. Dot plots represent cell proliferation after treatment with dAmBdpTP (A), dUmBdpTP (B) or dCmBdpTP (C) in presence of transporter SNTT. Standard EdU cell proliferation assay is also included (D) for comparison. Relative cell DNA content was detected by DAPI counterstaining (x axis); y axis corresponds to the fluorescence of a fluorophore covalently bounded to DNA. As the cell proliferates its DNA relative content (x axis) increases from 100% (in G0/G1 phase of cell cycle; bottom left) to 200% (in G2/M phase; bottom right) during S phase (middle up) when new DNA is synthesized. Experiments with dNmBdpTP (A-C) were measured in the same scale, EdU incorporation (D) was detected by Cy3 fluorophore, i.e. different conditions and scale were used; note the logarithmic scale of y-axis. 13 ACS Paragon Plus Environment


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In order to verify covalent labelling of DNA through incorporation of dNmBdp nucleotides and rule out any possible non-covalent staining (e.g. through intercallation), we performed the flow cytometric analysis (Fig. 5) analogous to the EdU cell proliferation assay. Experiments with all tested dNmBdpTPs displayed the horseshoe pattern (the region of dot plot associated with the S phase is moved upward on the y axis) which unequivocally proves the incorporation of the fluorophore into DNA (Figure 5A-D), similarly as in standard treatment with BrdU and antibody.53,54 The order of incorporation efficiency found by cytometry (height of the horseshoe pattern) is in agreement with the confocal microscopy measurement (Fig. 5; dCmBdpTP > dUmBdpTP > dAmBdpTP). On the other hand, treatment with dUTP-Alexa-488 and SNTT did not provide characteristic horseshoe pattern indicating poor incorporation of the fluorophore into genomic DNA (Figure S28 in SI). Although so far the cytometry using direct incorporation of dCmBdpTP does not provide clear separation between particular cell phases, it is technically much simpler and does not require any additional operation (i.e. click reaction with fluorescent dye in case of EdU or treatment with antibody in case of BrdU53,54). There is clearly some space for further tuning of the experimental conditions in order to make this approach competitive for routine cell cycle analysis.

Conclusions Facile and straightforward synthesis of base-modified nucleosides and dNTPs bearing hexamethylated phenyl-bodipy fluorophore was developed. The title nucleosides dNmBdp and triphosphates dNmBdpTP displayed bright green fluorescence with very high quantum yields even in water. All dNmBdpTP were good substrates to DNA polymerases in PEX and were also applicable in PCR amplifications, though in 1:1 to 1:20 ratio with natural dNTPs but still producing strongly fluorescent PCR amplicons. Most importantly, the new dNmBdpTP were used in combination with recently reported32 transporter SNTT for fluorescent staining of genomic DNA in living cells. The best performing was the cytosine nucleotide dCmBdpTP which efficiently produced bright fluorescent labelling of chromatin in living cells and was even used for in cellulo 14 ACS Paragon Plus Environment

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live study of mitosis by confocal microscopy. All three modified dNmBdpTP were also successfully used for staining of DNA in flow cytometry without the need of use of any further external reagent or antibody. These dNmBdpTP, and in particular the dCmBdpTP, perform much better than commercial dUTP-Alexa-48848,49 in DNA labelling in live cells and certainly have potential for further use in super-resolution microscopy of chromatin,55 staining of mitochondrial DNA and other attractive applications. Studies along these lines are under way in our labs.

ASSOCIATED CONTENT Supporting Information Available. Full experimental part, characterization of compounds, copies of spectra, additional figures and a video of the mitosis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Michal Hocek: Phone: +420 220183324. E-mail: [email protected] ORCID Michal Hocek: 0000-0002-1113-2047

Acknowledgement: Funding by Czech Science Foundation (206-12-G151 to P.G.G. and M.H. and 17-14791S to Z.Z. and T.K.), by the Czech Academy of Sciences (Praemium Academiae award to M.H.) and by the European Regional Development Fund; OP RDE (No. CZ.02.1.01/0.0/0.0/16_019/0000729) is gratefully acknowledged.

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