Intermolecular dark resonance energy transfer (DRET): Upgrading fluorogenic DNA sensing

The sensitivity of FRET-based sensing is usually limited by the spectral overlaps of the FRET donor and acceptor, which generate a poor signal-to-noise ratio. To overcome this limitation, a quenched donor presenting a large Stokes shift can be combined with a bright acceptor to perform Dark Resonance Energy Transfer (DRET). The consequent fluorogenic response from the acceptor considerably improves the signal-to-noise ratio. To date, DRET has mainly relied on a donor that is covalently bound to the acceptor. In this context, our aim was to develop the first intermolecular DRET pair for specific sensing of nucleic acid sequences. To this end, we designed DFK, a push-pull probe based on a fluorenyl π-platform that is strongly quenched in water. DFK was incorporated into a series of oligonucleotides and used as a DRET donor with Cy5-labeled complementary sequences. In line with our expectations, excitation of the dark donor in the double-labeled duplex switched on the far-red Cy5 emission and remained free of cross-excitation. The DRET mechanism was supported by time-resolved fluorescence measurements. This concept was then applied with binary probes, which confirmed the distance dependence of DRET as well as its potency in detecting sequences of interest with low background noise

Intermolecular resonance energy transfer from a quenched donor (DRET) : applications to fluorogenic detection of nucleic acids and their visualisation

Les récentes découvertes de l'implication des acides nucléiques dans un large éventail de processus biologiques ont révélé l'importance de pouvoir les détecter et les localiser précisément au sein des cellules. Pour y parvenir, de nombreuses stratégies ont été élaborées. Le processus de transfert d'énergie par résonance entre deux molécules fluorescentes (FRET) a largement contribué à relever ce défi. Cependant, des problèmes liés aux recouvrements spectraux du donneur et de l'accepteur limitent sa sensibilité. Des chercheurs ont récemment découvert la possibilité de réaliser un transfert d'énergie par résonance à partir d'un donneur éteint (Dark-RET : DRET). De nouveaux fluorophores, combinant un donneur éteint et un accepteur fluorescent au sein de la même entité moléculaire, ont été conçus. En détournant ce concept au travers d'une approche intermoléculaire, nous avons développé une nouvelle méthode de détection fluorogénique des acides nucléiques. Celle-ci présente l'avantage de réduire considérablement le bruit de fond de manière très simple. Le fluorène push-pull DFK, qui remplit parfaitement toutes les exigences d'un donneur éteint pour du DRET intermoléculaire, a été synthétisé sous forme d'un amidite. Ce dernier a été incorporé dans de nombreuses sondes d'acide nucléique. L'hybridation de l'une d'entre elles avec sa séquence complémentaire marquée par un accepteur brillant a généré un facteur d'amplification record de 256. Ce résultat a confirmé la capacité du DRET à conférer à un donneur éteint le potentiel de fluorescence de l'accepteur avec lequel il est associé. La preuve de concept a ensuite été réalisée in cellulo pour la détection de la séquence ARNm oskar. Le signal de fluorescence correspondant à sa localisation est alors sorti très nettement du bruit de fond résiduel. Néanmoins, les performances de cette nouvelle méthode de détection des acides nucléiques varient fortement en fonction de la composition nucléotidique des séquences. Afin de la rendre universelle, il est essentiel de se concentrer sur la conception de nouveaux fluorophores, insensibles à cette variation. Le développement de tels outils fluorogéniques permettrait d'éclairer de nombreux processus biologiques sous les feux d'un autre projecteur.Recent discoveries of the involvement of nucleic acids in a wide range of biological processes have highlighted the importance of being able to detect and localise them precisely within cells. To achieve this, many strategies have been developed. The process of resonance energy transfer between two fluorescent molecules (FRET) has made a major contribution to meeting this challenge. However, problems related to spectral overlaps between the donor and acceptor limit its sensitivity. Researchers have recently discovered the possibility of performing resonance energy transfer from a quenched donor (Dark-RET: DRET). New fluorophores, combining a quenched donor and a fluorescent acceptor within the same molecular entity, have been designed. By revisiting this concept through an intermolecular approach, we have developed a new method for fluorogenic detection of nucleic acids. This has the advantage of considerably reducing the background noise in a very simple way. The push-pull fluorophore DFK, which fulfils all the requirements of a quenched donor for intermolecular DRET, has been synthesized as an amidite. The latter has been incorporated into numerous nucleic acid probes. Hybridisation of one of these with its complementary sequence labelled with a bright acceptor generated a record amplification factor of 256. This result confirmed the ability of DRET to confer on a quenched donor, the fluorescence potential of the acceptor with which it is associated. The proof of concept was then performed in cellulo for the detection of the oskar mRNA sequence. The fluorescence signal corresponding to its localisation then emerged very clearly from the residual background. Nevertheless, the performance of this new nucleic acid detection method varies greatly depending on the nucleotide composition of the sequences. In order to make it universal, it is essential to focus on the design of new fluorophores, insensitive to this variation. The development of such fluorogenic tools would allow many biological processes to be illuminated under a different spotlight offering a new angle of examination

Transfert d'énergie intermoléculaire par résonance à partir d'un donneur éteint (DRET) : applications à la détection fluorogénique des acides nucléiques et leur visualisation

Recent discoveries of the involvement of nucleic acids in a wide range of biological processes have highlighted the importance of being able to detect and localise them precisely within cells. To achieve this, many strategies have been developed. The process of resonance energy transfer between two fluorescent molecules (FRET) has made a major contribution to meeting this challenge. However, problems related to spectral overlaps between the donor and acceptor limit its sensitivity. Researchers have recently discovered the possibility of performing resonance energy transfer from a quenched donor (Dark-RET: DRET). New fluorophores, combining a quenched donor and a fluorescent acceptor within the same molecular entity, have been designed. By revisiting this concept through an intermolecular approach, we have developed a new method for fluorogenic detection of nucleic acids. This has the advantage of considerably reducing the background noise in a very simple way. The push-pull fluorophore DFK, which fulfils all the requirements of a quenched donor for intermolecular DRET, has been synthesized as an amidite. The latter has been incorporated into numerous nucleic acid probes. Hybridisation of one of these with its complementary sequence labelled with a bright acceptor generated a record amplification factor of 256. This result confirmed the ability of DRET to confer on a quenched donor, the fluorescence potential of the acceptor with which it is associated. The proof of concept was then performed in cellulo for the detection of the oskar mRNA sequence. The fluorescence signal corresponding to its localisation then emerged very clearly from the residual background. Nevertheless, the performance of this new nucleic acid detection method varies greatly depending on the nucleotide composition of the sequences. In order to make it universal, it is essential to focus on the design of new fluorophores, insensitive to this variation. The development of such fluorogenic tools would allow many biological processes to be illuminated under a different spotlight offering a new angle of examination.Les récentes découvertes de l'implication des acides nucléiques dans un large éventail de processus biologiques ont révélé l'importance de pouvoir les détecter et les localiser précisément au sein des cellules. Pour y parvenir, de nombreuses stratégies ont été élaborées. Le processus de transfert d'énergie par résonance entre deux molécules fluorescentes (FRET) a largement contribué à relever ce défi. Cependant, des problèmes liés aux recouvrements spectraux du donneur et de l'accepteur limitent sa sensibilité. Des chercheurs ont récemment découvert la possibilité de réaliser un transfert d'énergie par résonance à partir d'un donneur éteint (Dark-RET : DRET). De nouveaux fluorophores, combinant un donneur éteint et un accepteur fluorescent au sein de la même entité moléculaire, ont été conçus. En détournant ce concept au travers d'une approche intermoléculaire, nous avons développé une nouvelle méthode de détection fluorogénique des acides nucléiques. Celle-ci présente l'avantage de réduire considérablement le bruit de fond de manière très simple. Le fluorène push-pull DFK, qui remplit parfaitement toutes les exigences d'un donneur éteint pour du DRET intermoléculaire, a été synthétisé sous forme d'un amidite. Ce dernier a été incorporé dans de nombreuses sondes d'acide nucléique. L'hybridation de l'une d'entre elles avec sa séquence complémentaire marquée par un accepteur brillant a généré un facteur d'amplification record de 256. Ce résultat a confirmé la capacité du DRET à conférer à un donneur éteint le potentiel de fluorescence de l'accepteur avec lequel il est associé. La preuve de concept a ensuite été réalisée in cellulo pour la détection de la séquence ARNm oskar. Le signal de fluorescence correspondant à sa localisation est alors sorti très nettement du bruit de fond résiduel. Néanmoins, les performances de cette nouvelle méthode de détection des acides nucléiques varient fortement en fonction de la composition nucléotidique des séquences. Afin de la rendre universelle, il est essentiel de se concentrer sur la conception de nouveaux fluorophores, insensibles à cette variation. Le développement de tels outils fluorogéniques permettrait d'éclairer de nombreux processus biologiques sous les feux d'un autre projecteur

Hybrid Gold(I) NHC-Artemether Complexes to Target Falciparum Malaria Parasites

The emergence of Plasmodium falciparum parasites, responsible for malaria disease, resistant to antiplasmodial drugs including the artemisinins, represents a major threat to public health. Therefore, the development of new antimalarial drugs or combinations is urgently required. In this context, several hybrid molecules combining a dihydroartemisinin derivative and gold(I) N-heterocyclic carbene (NHC) complexes have been synthesized based on the different modes of action of the two compounds. The antiplasmodial activity of these molecules was assessed in vitro as well as their cytotoxicity against mammalian cells. All the hybrid molecules tested showed efficacy against P. falciparum, in a nanomolar range for the most active, associated with a low cytotoxicity. However, cross-resistance between artemisinin and these hybrid molecules was evidenced. These results underline a fear about the risk of cross-resistance between artemisinins and new antimalarial drugs based on an endoperoxide part. This study thus raises concerns about the use of such molecules in future therapeutic malaria policies

Hybrid Gold(I) NHC-Artemether Complexes to Target Falciparum Malaria Parasites

International audienceThe emergence of Plasmodium falciparum parasites, responsible for malaria disease, resistant to antiplasmodial drugs including the artemisinins, represents a major threat to public health. Therefore, the development of new antimalarial drugs or combinations is urgently required. In this context, several hybrid molecules combining a dihydroartemisinin derivative and gold(I) N-heterocyclic carbene (NHC) complexes have been synthesized based on the different modes of action of the two compounds. The antiplasmodial activity of these molecules was assessed in vitro as well as their cytotoxicity against mammalian cells. All the hybrid molecules tested showed efficacy against P. falciparum, in a nanomolar range for the most active, associated with a low cytotoxicity. However, cross-resistance between artemisinin and these hybrid molecules was evidenced. These results underline a fear about the risk of cross-resistance between artemisinins and new antimalarial drugs based on an endoperoxide part. This study thus raises concerns about the use of such molecules in future therapeutic malaria policies

Artemisinin-Derivative-NHC-gold(I)-Hybrid with Enhanced Cytotoxic Activity Through Inhibiting NRF2 Transcriptional Activity

A family of original bis(artemisinin-NHC)gold(I) complexes have been synthesized. These hybrid molecules combine two biological active motifs, an artemisinin derivative (DHA) and a cationic bis(NHC)gold(I) unit. One of these complexes, complex 2a, has been analyzed by single-crystal X-ray diffraction and tested in depth for its anticancer properties. Complex 2a shows strong anticancer activities on a representative panel of human cancer cell models from 8 different localizations (prostate, breast, lung, liver, bladder, bone, acute and chronic myeloid leukemias). Complex 2a shows anticancer activity to a much better degree than Auranofin and DHA standards with GI50 values in nM range, together with a superior selectivity in regard to non-cancer cell models. Next to expected ROS formation and TrxR inhibition, an original and distinctive mechanism of action through inhibition of NRF2 - a transcription factor strongly associated with aggressiveness and resistance to cancer therapies - with an IC50 value at nM range has been evidenced. Importantly, the NRF2 inhibitory effect of complex 2a could remarkably sensitize to sorafenib in HepG2 liver cells, in which dysregulated NRF2 signaling is known to be associated with primary and acquired drug resistance. Moreover, complex 2a also inhibited NF-κB and HIF transcriptional activities, which are also linked to progression and resistance in cancer. Our findings provide experimental evidence that hybrid (NHC)gold(I) molecules - such as complex 2a - represent a new class of organometallic hybrid molecules that may yield new therapeutic agents.</div

The therapeutic potential of HIF-2 antagonism in renal cell carcinoma

International audienceA family of original bis(artemisinin-NHC)gold(I) complexes have been synthesized. These hybrid molecules combine two biological active motifs, an artemisinin derivative (DHA) and a cationic bis(NHC)gold(I) unit. One of these complexes, complex 2a, has been analyzed by single-crystal X-ray diffraction and tested in depth for its anticancer properties. Complex 2a shows strong anticancer activities on a representative panel of human cancer cell models from 8 different localizations (prostate, breast, lung, liver, bladder, bone, acute and chronic myeloid leukemias). Complex 2a shows anticancer activity to a much better degree than Auranofin and DHA standards with GI50 values in nM range, together with a superior selectivity in regard to non-cancer cell models. Next to expected ROS formation and TrxR inhibition, an original and distinctive mechanism of action through inhibition of NRF2 - a transcription factor strongly associated with aggressiveness and resistance to cancer therapies - with an IC50 value at nM range has been evidenced. Importantly, the NRF2 inhibitory effect of complex 2a could remarkably sensitize to sorafenib in HepG2 liver cells, in which dysregulated NRF2 signaling is known to be associated with primary and acquired drug resistance. Moreover, complex 2a also inhibited NF-κB and HIF transcriptional activities, which are also linked to progression and resistance in cancer. Our findings provide experimental evidence that hybrid (NHC)gold(I) molecules - such as complex 2a - represent a new class of organometallic hybrid molecules that may yield new therapeutic agents.</div

Artemisinin-Derivative-NHC-gold(I)-Hybrid with enhanced cytotoxic activity through inhibiting NRF2 transcriptional activity

International audienceA family of hybrid complexes combining two biologically active motifs, an artemisinin derivative and a cationic bis(NHC)‐gold(I) unit, has been synthesized. One of these complexes, 2 a, has been analyzed by single‐crystal X‐ray diffraction. 2 a shows strong anticancer activities on a large panel of human cancer cell models (prostate, breast, lung, liver, bladder, bone, acute and chronic myeloid leukemias) with GI50 values in the nm range, together with a high selectivity. An original and distinctive mechanism of action, that is, through inhibition of the redox antioxidant NRF2 transcription factor (strongly associated with aggressiveness and resistance to cancer therapies) has been evidenced. 2 a could remarkably sensitize to sorafenib in HepG2 liver cells, in which dysregulated NRF2 signaling is linked to primary and acquired drug resistance. 2 a also inhibited NF‐κB and HIF transcriptional activities, which are also associated with progression and resistance in cancer. Our findings provide evidence that hybrid (NHC)gold(I) compounds represent a new class of organometallic hybrid molecules that may yield new therapeutic agents

Control of Intermolecular Photoinduced Electron Transfer in Deoxyadenosine‐Based Fluorescent Probes

International audienceIn this paper, we report on the Photoinduced Electron Transfer (PET) reaction between a donor (adenine analog) and an acceptor (3-methoxychromone derivative) in the context of designing efficient fluorescent probes as DNA sensors. Firstly, Gibbs energy was investigated in disconnected donor-acceptor systems by Rehm-Weller equation. The oxidation potential of the adenine derivative was responsible for exergonicity of the PET reaction in separated combinations. Then, the PET reaction in donor-π-acceptor conjugates was investigated using steady-state fluorescence spectroscopy, acid-mediated PET inhibition and transient absorption techniques. In conjugated systems, PET is a favorable pathway of fluorescent quenching when an electron-rich adenine analog (d7A) was connected to the fluorophore (3MC). We found that formation of ground-state complexes even at nM concentration range dominated the dye photophysics and generated poorly emissive species likely through intermolecular PET from d7A to 3MC. On the other hand, solution acidification disrupts complexation and turns on dye emission. Bridging an electron-poor adenine analog with high oxidation potential (8d7A) to 3MC presenting low reduction potential is another alternative to prevent complex formation and produce highly emissive monomer conjugates