RNase H-Assisted Imaging of Peroxynitrite in Living Cells with 5

Aug 4, 2016 - In the present study, we demonstrate that 5′-boronic acid-modified oligonucleotides could inhibit RNase H activity; actvity which coul...
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RNase H-Assisted Imaging of Peroxynitrite in Living Cells with 5’-Boronic Acid Modified DNA Maeva Reverte, Anais Vaissiere, Prisca Boisguerin, Jean-Jacques Vasseur, and Michael Smietana ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00401 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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RNase H-Assisted Imaging of Peroxynitrite in Living Cells with 5’Boronic Acid Modified DNA Maëva Reverte,† Anaïs Vaissiere,‡ Prisca Boisguerin,‡ Jean-Jacques Vasseur, † and Michael Smietana*,† †

Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Université de Montpellier, ENSCM, Place Bataillon, CC1704 34095 Montpellier, France ‡ Centre de Recherche de Biologie Cellulaire de Montpellier, UMR 5267 CNRS, 1919 Route de Mende, 34293 Montpellier, France Supporting Information Placeholder ABSTRACT: RNase H is a ubiquitous intracellular enzyme able to selectively hydrolyse the RNA strand of an RNA/DNA hybrid duplex. In the present study, we demonstrate that 5’-boronic acidmodified oligonucleotides could inhibit RNase H activity; actvity which could subsequently be restored upon oxidation of the boronic acid moiety. This unprecedented dual property was exploited to engineer a new stimuli-responsive system for the chemoselective imaging of endogenous and exogenous peroxynitrite in RAW264.7 cells.

KEYWORDS: RNase H, boronic acid, peroxynitrite, DANN, stimuli-responsive Reactive Oxygen Species (ROS), are highly reactive oxygencontaining oxidants that play fundamental regulatory roles of many physiological processes.1-4 Among these species, peroxynitrite (ONOO-), a potent oxidant and nitrating agent produced in cellulo by spontaneous reaction between nitric oxide (NO) and superoxide (O2•-) causes oxidative damages to cellular proteins,5 lipids6,7 and nucleic acids.8,9 As a result, peroxynitrite has been implicated as a key metabolite in numerous disorders including Alzheimer’s disease, neurodegenerative diseases, cancer or inflammatory phenomenon.10-13 Therefore, molecular imaging of this oxygen metabolite is an important prerequisite to study its biological impact and to screen for peroxynitrite capturing compounds both in vitro and in cellulo. Accordingly, a few synthetic fluorescent probes have emerged as powerful tools to detect intracellular oxidants such as ONOO-.14-26 The use of fluorescent arylboronates, which undergo peroxynitrite-mediated oxidation, appeared particularly attractive.20,27-29 Nevertheless, a recurrent issue about these systems concerns their chemoselectivity and water-solubility. Indeed while ONOO- reacts with arylboronate derivatives nearly a million times faster than hydrogen peroxide, interferences by other ROS might be observed especially with long incubation time.9,30 Recently, Ai et al. introduced a boronic acid moiety on a genetically encoded fluorescent protein for selective detection of peroxynitrite in cells,19,31 but to the best of our knowledge an enzyme-assisted fluorescent detection of cellular peroxynitrite using boronic acid based probes has never been reported. Our group has been particularly interested in modifying nucleic acids at their 5’ extremity with 5’-boronoisosteric analogues of nucleotide 5’-monophosphates. These modified sequences have

notably been employed to engineer reversible boronate internucleosidic linkages with 3’-ribonucleotidic partners under enzymefree and activator-free conditions.32-37 Recently, we evaluated 5’ended boronic acid-modified oligonucleotides against various nucleases at the single and double stranded levels. Our results revealed that this modification was able to improve the stability of the modified probe but also protect its complementary strand from degradation.38 This unique property was eventually used to detect single point mutations assisted by Snake Venom Phosphodiesterase. In continuation to our work, we became interested in ribonuclease H (RNase H). RNase H is a ubiquitous intracellular enzyme able to specifically degrade the RNA strand of a RNA/DNA hybrid duplex.39 Eliciting RNase H activity is of prime importance in antisense strategies involving chemically modified oligonucleotides sequences (AONs).40 In most of the cases, fully modified AONs fail to activate RNase H, but the synthesis of chimeric oligonucleotides having 3’- and 5’-modifications along with a normal phosphodiester central core have been employed to protect the AONs from exonucleases while maintaining RNase H activity.41 By contrast, the pivotal role of RNase H in HIV replication has prompted the development of RNase H inhibitors.42 Herein we report the exceptional ability of 5’-boronic acid modified oligonucleotides to inhibit RNase H activity and the use of intracellular RNase H for imaging exogenous and endogenous ONOO- in RAW264.7 murine macrophages (Figure 1).

Figure 1. Schematic representation of the sensing of peroxynitrite with 5’-boronic acid modified oligonucleotides in the presence of RNase H.

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Our first set of experiments involved the synthesis of ODN1 (5′-TbnGAATACAAATT-3′), a 12mer DNA sequence modified at the 5’ extremity by a boronic acid function able to hybridize a 21mer RNA strand ORN1 (5′UUUGTAUUCAGCCCAUATCUU-3′). The resulting ODN1/ORN1 hybridization lead to a 12 bp duplex with the boronic modification at its center (Figure 2). The natural analogue ODN2 (5′-TGAATACAAATT-3′) was also prepared for comparison purposes. ODN1/ORN1 and ODN2/ORN1 were then incubated in the presence of 10 U of E. coli RNase H. Native PAGE analysis reported in Figure 2 demonstrate that while the natural duplex elicits RNase H hydrolysis of the RNA target in less than 15 min, duplex ODN1/ORN1 remains intact even after 24 h of incubation. The unexpected ability of 5’-boronic acid to inhibit RNase H activity was further demonstrated with modified sequences as short as 6mers (see Supporting Information). However, any attempt made with sequences modified at their 5’-extremity by a phenyl boronic acid or a triazoylmethane boronic acid failed to inhibit RNase H activity thus highlighting the importance of the bioisosteric boronothymidine (data not shown).

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(NO2-), and nitrate (NO3-) in a commercial RNase H buffer at pH 8,3. At 100 µM ClO- elicited a fluorescence enhancement of (II0)/I = 0.5 after 15 min of incubation while the ONOO- response was 3 times higher ((I-I0)/I = ca. 1.5) (Figure 3A). The selectivity of ONOO- was further confirmed when the concentration of each species was reduced to 10 µM. Notably, ONOO- was the only oxidizing reagent able to induce a fluorescent enhancement after 15 min of incubation ((I-I0)/I = 0.52, Figure 3B). More importantly, even after 60 min of incubation ONOO- induced a large fluorescence increase ((I-I0)/I = ca. 0,75) while all other tested reagent triggered no fluorescence response (Figure 3C). Considering that longer incubation time is often accompanied by the interference of other ROS, these results demonstrate the high specificity of peroxynitrite to restore RNase H activity, an essential property for cellular applications.

Figure 3. Fluorescence responses of ODN3/ORN2 (2 µM) in the presence of RNase H (10 U) and various oxygen species at different incubation time. A) 100 µM, 15 min and B) 10 µM, 15 min. C) 10 µM, 60 min. λex = 498 nm and λem = 518 nm. (Ex slit: 5.0 nm, Em slit: 10.0 nm).

Figure 2. Native PAGE of ODN1/ORN1 (5’TbnGAATACAAATT-3’/5’UUUGTAUUCAGCCCAUATCUU-3’), and ODN2/ORN1 (5’TGAATACAAATT-3/5’-UUUGTAUUCAGCCCAUATCUU-3’ in the presence of RNase H (10 U) at different times. These results prompted us to evaluate the ability of these aliphatic boronic acids to be oxidized to the corresponding homologated alcohol and ultimately if this resulting modification would alter RNase H activity. As stimuli-responsive fluorescent probes are receiving considerable attention for bioimaging,43-45 we designed probes bearing the dye molecule 6-FAM at the 5’-end of the RNA target ORN2 (5’-FAMUUUGUAUUCAGCCCAUAUCUU) and the quencher BHQ-1 at the 3’ end of ODN3, a complementary 12mer modified at its 5’end by a boronic acid function (5’-TbnGAATACAAATT-3’BHQ-1). Preliminary experiments performed in the absence of ONOO- and with 10 U of RNase H and 2 µM of the duplex showed only residual fluorescence over a period of 3h indicating an efficient fluorescence quenching and no RNase H activity. However upon treatment with 50 equiv (100 µM) of ONOO-, an intense fluorescence enhancement of (I-I0)/I = ca. 1.5 at 518 nm was observed after 15 min of incubation thus demonstrating that oxidation of the boronic acid moiety to the corresponding alcohol restored RNase H activity. With these results in hand, we next evaluated the response ability and the selectivity of the ODN3/ORN2 system toward a series of biologically relevant ROS including hydrogen peroxide (H2O2), hypochlorite (ClO-), nitrite

We next examined the fluorescence response of our system to different concentrations of ONOO- after 15 and 60 min of incubation. As shown in Figure 4 upon treatment with increasing concentration of ONOO-, the fluorescence increased progressively. Linear calibration curves of the fluorescence intensities vs. ONOO- concentration from 0.1 to 10 µM were obtained in both cases (R2 = 0.9898 and 0.9999) allowing to determine the limit of detection (LOD) to be 270 and 28 nM respectively based on SD/S = 3.3. Thus, longer incubation time increases sensitivity without affecting the selectivity.

Figure 4. Fluorescence spectra and intensity changes of ODN3/ORN2 (2µM) in the presence of RNase H (10 U) as a function of ONOO- concentration (0-10 µM) after A) 15 min and B) 60 min of incubation at 37°C. Inset: the corresponding linear relationship between fluorescent intensity and ONOO- concentration (0.1-10 µM). λex = 498 nm and λem = 518 nm (Ex slit: 3.0 nm, Em slit: 10.0 nm). Finally, we evaluated the capacity of our system for intracellular imaging of peroxynitrite. RAW264.7 murine macrophages are known to produce ONOO- via immunogenic stimuli with interferon–γ (IFN-γ) and lipopolysaccharide (LPS).46 RAW264.7 cells transfected with duplex ODN3/ORN2 (150 nM) were found to

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have no fluorescence as observed by epifluorescence microscopy at 498 nm even after 5 h at 37 °C (Figure 5B). When external peroxynitrite (1 µM) was added to the transfected cells, a high increase of fluorescence was observed after 5 minutes at 37°C, revealing that the system associated with the ubiquitous RNase H could monitor exogenous ONOO- in the cellular environment at low concentration and at physiological pH (Figure 5, C).47 Moreover, upon stimulation of RAW264.7 cells with IFN-γ and LPS for 12 hours, and transfection of ODN3/ODN2 a dramatic fluorescence enhancement was observed (Figure 5D). Control experiments performed with stimulated cells pretreated with aminoguanidine (NOS inhibitor) or TEMPO (scavenger of peroxynitrite) and then transfected with ODN3/ODN2 led to no fluorescence emission even after 5h at 37°C (Figures 5E and 5F). Overall, the strong fluorescence observed from activated RAW 264.7 cells confirmed the ability of our system to detect endogenous peroxynitrite in living cells at physiologically relevant concentrations.

living cells is expected to enable new development for the sensing of ROS species but also trigger new biotechnological researches.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis and analysis of Oligonucleotides, detailed procedures and fluorescence spectra of RNase H-based experiments (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We acknowledge the Agence Nationale de la Recherche (ANR “PRODYGY”–11-JS07-005-01), the Fondation de la Recherche Medicale (DBS 20140930769), the CNRS and the Région Languedoc-Roussillon (Programme “Chercheur d’Avenir”) for financial support. We thank Dr K. Konate for assistance with transfection experiments.

REFERENCES

Figure 5. Fluorescence images of RAW264.7 murine macrophages under different conditions. A) RAW264.7 cells only; B) Cells transfected with ODN3/ORN1 (150 nM, 5h, 37°C); C) Cells transfected with ODN3/ORN1 (150 nM) and treated with ONOO(1 µM, 5 min, 37°C); D) Cells stimulated with LPS (1 µg.mL-1) and IFN-γ (50 ng.mL-1) for 12 h, and then transfected with ODN3/ORN1 (150 nM, 5 h, 37°C). E) Cells pretreated with AG (5 mM) during LPS (1 µg.mL-1)/IFN-γ (50 ng.mL-1) stimulation for 12 h, and then transfected with ODN3/ORN1 (150 nM, 5 h, 37°C). F) Cells pretreated with TEMPO (300 µM) during LPS (1 µg.mL-1)/IFN-γ (50 ng.mL-1) stimulation for 12 h, and then transfected with ODN3/ORN1 (150 nM, 5h, 37°C). n = 2 for each condition. In summary, we report herein a new intracellular sensing strategy based on the ability of oligodeoxynucleotides modified by a boronic acid to inhibit RNase H activity. This unprecedented property was exploited for the development of a selective and sensitive RNase H–assisted imaging of intracellular peroxynitrite. Oxidation of the boronic acid to the corresponding alcohol by ONOO- restored RNase H activity and triggered the degradation of the labelled RNA sequence visualized by a significant fluorescent increase. Considering the critical role of RNase H in a wide variety of cellular processes, the ability of bioisosteric 5’borononucleic acids modified sequences to inhibit RNase H in

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For TOC only 5’-End boronic acid-modified oligonucleotides have been found to inhibit RNase H, a non-sequence-specific endonuclease that catalyze the cleavage of the RNA strand of RNA/DNA hybrids. This unique property has been exploited for the selective detection and imaging of peroxynitrite in living cells.

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