Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags.

Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering.

Protein-protein interactions (PPIs) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of splitFAST2, an improved reporter displaying higher brightness, lower self-complementation, and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, splitFAST2 is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

Engineering of Tunable Allosteric-like Fluorogenic Protein Sensors.

Optical protein sensors that enable detection of relevant biomolecules of interest play central roles in biological research. Coupling fluorescent reporters with protein sensing units has enabled the development of a wide range of biosensors that recognize analytes with high selectivity. In these sensors, analyte recognition induces a conformational change in the protein sensing unit that can modulate the optical signal of the fluorescent reporter. Here, we explore various designs for the creation of tunable allosteric-like fluorogenic protein sensors through incorporation of sensing protein units within the chemogenetic fluorescence-activating and absorption-shifting tag (FAST) that selectively binds and stabilizes the fluorescent state of 4-hydroxybenzylidene rhodanine (HBR) analogs. Conformational coupling allowed us to design analyte-responsive optical protein sensors through allosteric-like modulation of fluorogen binding.

Vaccination acceptability in the French general population and related determinants, 2000-2021.

BACKGROUND: This study describes the evolution of vaccination acceptability and associated determinants in the French general population between 2000 and 2021, and vaccinations with the highest vaccine hesitancy between 2010 and 2021. METHODS: Data were collected from the nine national 'Health Barometer' cross-sectional surveys conducted between 2000 and 2021. These surveys included French-speaking individuals aged 18-75 years old who were selected through randomly generated landline and mobile phone numbers. Participants were asked about their acceptability of vaccination in general and their vaccine hesitancy toward any particular vaccinations. Determinants of vaccination acceptability were studied using univariate and multivariate Poisson regressions. RESULTS: The proportion of persons who found vaccination acceptable in general (i.e., answering "very" or "somewhat" favourable in the survey interview) decreased from 91.1% in 2000 to 61.2% in 2010 (the latter year coinciding with the 2009 H1N1 influenza pandemic), increased in 2014 (78.8%), slightly fluctuated until 2019 (74.2%), and increased again in both 2020 (80.0%) and 2021 (82.5%) during the COVID-19 pandemic. Irrespective of the year, acceptability was higher among persons with higher incomes, those with a higher education level, and individuals not living alone. In 2021, for the first time, vaccination acceptability was higher among persons over 44 years old (versus 18-24 year-olds) and among retired persons (versus workers). The highest hesitancy rate for a vaccine was for the 2009 H1N1 influenza virus in 2010 (41% answering "somewhat" or "very" unfavourable). In 2021, the highest rate was for the COVID-19 vaccine (21%). DISCUSSION: Unlike the experience of the 2009 AH1N1 influenza pandemic, which led to a collapse in vaccination acceptability among the French general population, acceptability continued to increase during the COVID-19 pandemic. However, the pre-2010 level was not reached. Our results show a tendency towards a widening social and economic gap in terms of vaccine acceptability over time.

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

Molecular tools enabling the control and observation of the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allows the design of a new class of biosensors for the study of processes such as signal transduction and apoptosis.

A fluorescent reporter system for anaerobic thermophiles.

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli-Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes.

Cage-like structures based on constrained cyclic arylopeptoids.

The access to cupola-like or tube-like structures from ortho- and meta-arylopeptoid macrocycles was explored through CuAAC reaction using a partially flexible bis(azide) and CuI-N-heterocyclic carbene as catalyst. NMR studies showed that a bis-triazolium bicylic compound in the ortho-series adopts well-defined structure in polar aprotic and protic solvents. Besides, preliminary study revealed its potential for oxoanion recognition.

Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles.

A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.

SARS-CoV-2 testing, infection and places of contamination in France, a national cross-sectional study, December 2021.

BACKGROUND: This study aimed to describe the use of diagnostic testing for SARS-CoV-2 in France until December 2021, the characteristics of people infected, and places of contamination. METHODS: Data were collected from the national 2021 Health Barometer cross-sectional study, which was conducted between February and December 2021 and included French-speaking individuals aged 18-85 years old selected through randomly generated landline and mobile phone numbers. Participants were interviewed about COVID-19-like symptoms in the previous 12 months, diagnostic testing for SARS-CoV-2, positive diagnosis for SARS-CoV-2, and the place(s) of contamination. Determinants of diagnostic testing and of infection were studied using univariate and multivariate Poisson regressions. RESULTS: A total of 24,514 persons participated in the study. We estimated that 66.4% [65.0-67.7] of persons had been tested for SARS-CoV-2 the last time they experienced COVID-19-like symptoms, and that 9.8% [9.3-10.3] of the population in France - with or without symptoms - had been tested positive. Diagnostic testing was less frequent in men, unemployed persons, and people living alone; it was also less frequent during the first months of the pandemic. The estimated proportion of the population infected was higher in healthcare professionals (PRa: 1.5 [1.3-1.7]), those living in large cities ( > = 200 000 inhabitants, and Paris area) (1.4 [1.2-1.6]), and in households comprising > 3 persons (1.7 [1.5-2.0]). It was lower in retired persons (0.8 [0.6-0.97]) and those over 65 years old (0.6 [0.4-0.9]). Almost two-thirds (65.7%) of infected persons declared they knew where they were contaminated; 5.8% [4.5-7.4] reported being contaminated outdoors, 47.9% [44.8-51.0] in unventilated indoor environments, and 43.4% [40.3-46.6] in ventilated indoor environments. Specifically, 51.1% [48.0-54.2] declared they were contaminated at home or in a family of friend's house, 29.1% [26.4-31.9] at their workplace, 13.9% [11.9-16.1] in a healthcare structure, and 9.0% [7.4-10.8] in a public eating place (e.g., cafeteria, bar, restaurant). CONCLUSIONS: To limit viral spread, preventive actions should preferentially target persons tested least frequently and those at a higher risk of infection. They should also target contamination in households, healthcare structures, and public eating places. Importantly, contamination is most frequent in places where prevention measures are most difficult to implement.

Acceptability of mandatory vaccination against influenza, measles, pertussis and varicella by workers in healthcare facilities: a national cross-sectional study, France, 2019.

BACKGROUND: Vaccination of healthcare workers (HCW) aims to protect them and to reduce transmission to susceptible patients. Influenza, measles, pertussis, and varicella vaccinations are recommended but not mandatory for HCW in France. Insufficient vaccine coverage for these diseases in HCW has raised the question of introducing mandatory vaccination. We conducted a survey to estimate acceptability of mandatory vaccination for these four vaccines by HCW working in healthcare facilities (HCF) in France, and to identify associated determinants. METHODS: In 2019, we performed a cross-sectional survey of physicians, nurses, midwives and nursing assistants working in HCF in France using a randomised stratified three-stage sampling design (HCF type, ward category, HCW category). Data were collected in face-to-face interviews using a tablet computer. We investigated the possible determinants of acceptability of mandatory vaccination using univariate and multivariate Poisson regressions, and estimated prevalence ratios (PR). RESULTS: A total of 8594 HCW in 167 HCF were included. For measles, pertussis, and varicella, self-reported acceptability of mandatory vaccination (very or quite favourable) was 73.1% [CI95%: 70.9-75.1], 72.1% [69.8-74.3], and 57.5% [54.5-57.7], respectively. Acceptability varied according to i) HCW and ward category for these three vaccinations, ii) age group for measles and pertussis, and iii) sex for varicella. For mandatory influenza vaccination, acceptability was lower (42.7% [40.6-44.9]), and varied greatly between HCW categories (from 77.2% for physicians to 32.0% for nursing assistants). CONCLUSION: HCW acceptability of mandatory vaccination was high for measles, pertussis and varicella but not as high for influenza. Vaccination for COVID-19 is mandatory for HCW in France. Replication of this study after the end of the COVID-19 crisis would help assess whether the pandemic had an impact on their acceptability of mandatory vaccination, in particular for influenza.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Vaccination against influenza, measles, pertussis and varicella in workers in healthcare facilities in France: A national cross-sectional study in 2019.

BACKGROUND: Vaccine recommendations for healthcare workers (HCW) aim to protect them and reduce transmission to susceptible patients. We conducted a national randomised survey in 2019 whose main objectives were to estimate national vaccination coverage (VC) for measles, pertussis, varicella, and influenza in HCW working in healthcare facilities (HCF) in France, and to identify determinants associated with higher VC. METHODS: We performed a cross-sectional survey of physicians, nurses, midwives and nursing assistants in HCF using a random stratified three-stage sampling design. Data were collected during face-to-face interviews using a tablet computer and complemented with information from the individual HCW vaccination records. We investigated possible determinants of higher VC using univariate and multivariate Poisson regressions and estimated the prevalence ratio (PR). RESULTS: We included 8594 HCW working in 167 HCF. Self-declared VC was 73.3% (CI95%: 71.0-75.5) for measles in HCW with no history of measles (at least one dose), 53.5% (49.9-57.0) for pertussis (booster dose during adulthood), 26.4% (23.0-30.2) for varicella in HCW with no history of varicella (at least one dose) and 34.8% (32.8-37.4) for influenza. Taking into account the history of each disease and related VC, 14.6% and 10.1 % of HCW were susceptible to measles and varicella. VC varied by profession, age group, ward and sex. Higher influenza VC was observed in HCW working in wards where i) there was a staff vaccination contact person (PRa: 1.2, CI95% 1.1-1.4), ii) staff vaccination was organized in the ward (1.4: 1.2-1.6), iii) information on influenza vaccines was provided (1.2: 1.1-1.4), and iv) the ward manager supported the HCW vaccination campaign (1.3: 1.1-1.6). DISCUSSION: Over a 10-year period, VC for HCW working in HCF improved in France. However, vaccination objectives were not achieved for measles (95%) or influenza (80%). Vaccination efforts should be continued, especially in wards with at-risk patients.

Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing.

Fluorescent labels and biosensors play central roles in biological and medical research. Targeted to specific biomolecules or cells, they allow noninvasive imaging of the machinery that govern cells and organisms in real time. Recently, chemogenetic reporters made of organic dyes specifically anchored to genetic tags have challenged the paradigm of fully genetically encoded fluorescent proteins. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded tags, these chemogenetic reporters open new exciting prospects for studying cell biochemistry and biology. In this Account, we present the growing toolbox of fluorescence-activating and absorption-shifting tags (FASTs), small monomeric proteins of 14 kDa (125 amino acids residues) that can be used as markers to monitor gene expression and protein localization in live cells and organisms. Engineered by directed protein evolution from the photoactive yellow protein (PYP) from the bacterium Halorhodospira halophila, prototypical FAST binds and stabilizes the fluorescent state of live-cell compatible hydroxybenzylidene rhodanine chromophores. This class of chromophores are normally dark when free in solution or in cells because they dissipate light energy through nonradiative processes. The protein cavity of FAST allows the stabilization of the deprotonated state of the chromophore and blocks the chromophore into a planar conformation, which leads to highly fluorescent protein-chromophore assemblies. The use of such fluorogenic dyes (also called fluorogens) enables the imaging of FAST fusion proteins in cells with high contrast without the need to remove unbound ligands through separate washing steps. Fluorogens with various spectral properties exist nowadays allowing investigators to adjust the spectral properties of FAST to their experimental conditions. Molecular engineering allowed furthermore to generate membrane-impermeant fluorogens for the selective labeling of cell-surface proteins. Over the years, we generated a collection of FAST variants with expanded spectral properties or fluorogen selectivity using a concerted strategy involving molecular engineering and directed protein evolution. Moreover, protein engineering allowed us to adapt FASTs for the design of fluorescent biosensors. Circular permutation enabled the generation of FAST variants with increased conformational flexibility for the design of biosensors in which fluorogen binding is conditioned to the recognition of a given analyte. Bisection of FASTs into two complementary fragments allowed us furthermore to create split variants with reversible complementation that allow the detection and imaging of dynamic protein-protein interactions. We provide, here, a general overview of the current state of development of these different systems and their applications for advanced live cell imaging and biosensing and discuss potential future directions.

Are immigrants living in France more reluctant to receive vaccines than native-born French citizens? findings from the national health Barometer study.

BACKGROUND: France is one of the world's most vaccine hesitant countries and vaccine hesitancy (VH) is considered one of the world's leading threats to global health. However, little is known about VH in immigrant populations in France. Using data from the 2016 Health Barometer, we examined VH among newcomers, more established immigrants, and the native-born population in France. METHODS: Data was collected from French speaking individuals aged from 15 to 75 years old, residing in France. Individuals were selected through randomly generated landline and mobile phone numbers. Vaccine hesitancy was assessed through four questions and a "time spent in France" variable was created, using the year of arrival in France. Associations were studied using logistic regression. RESULTS: A sample of 15,216 participants residing in France included 1,524 foreign-born immigrants and 13,692 native-born individuals, with a mean age of 46-years. Most participants (75.7%) reported being favorable to vaccination regardless of country of origin but immigrants were less hesitant toward vaccinations than the host population. Foreign-born immigrants from North Africa had the most favorable views whereas those from sub-Saharan Africa held most unfavorable views on vaccination. With time spent in France, the opinions towards vaccination became more negative (aOR = 0.57, 95 %CI [0.40-0.79], p = 0.001) and the risk of vaccine refusal (aOR = 2.34, 95 %CI [1.45 - 3.78] p = 0.001) and reluctant acceptance of vaccines increased (aOR = 1.89 95 %CI [1.20 - 2.99], p = 0.006).Foreign-born individuals with the longest residency in France had more negative opinions than native-born individuals, regardless of region of origin. CONCLUSION: Immigrants were less hesitant toward vaccinations than the host population, but vaccine hesitancy increased with time spent in France. The provision of appropriate information and awareness to facilitate critical thinking towards antivaccine theories is necessary for immigrants in France.

Influenza vaccination coverage of professionals working in nursing homes in France and related determinants, 2018-2019 season: a cross-sectional survey.

BACKGROUND: The burden of influenza morbidity and mortality in nursing homes (NH) is high. Vaccination of residents and professionals working in NH is the main prevention strategy. Despite recommendations, vaccination coverage among professionals is generally low. METHODS: We performed a nationwide cross-sectional survey of NH using a single-stage stratified random sampling design to estimate influenza vaccination coverage in NH healthcare workers (HCW) and non-medical professionals in France during the 2018-2019 season, and to identify measures likely to increase it. For each NH, a questionnaire was completed with aggregated data by one member of the management team. A multivariate analysis was performed using a negative binomial regression. RESULTS: Five-hundred and eighty nine NH filled in the study questionnaire (response rate: 49.5%). When considering all professionals (i.e., HCW and non-medical professionals), overall vaccination coverage was 30.6% (95%CI [28.2-33.0], range: 1.6-96.2). Overall influenza vaccination coverage in HCW was 31.9% [29.7-34.1]. It varied according to occupational category: 75.5% [69.3-81.7] for physicians, 42.9% [39.4-46.4] for nurses, 26.7% [24.5-29.0] for nursing assistants, and 34.0% [30.1-38.0] for other paramedical personnel. Vaccination coverage was higher i) in private nursing homes (RRa: 1.3, [1.1-1.5]), ii) in small nursing homes (0.9 [0.8-0.9]), iii) when vaccination was offered free of charge (1.4, [1.1-1.8]), iv) when vaccination promotion for professionals included individual (1.6 [1.1-2.1]) or collective (1.3 [1.1-1.5]) information sessions, videos or games (1.4 [1.2-1.6], v) when information on influenza vaccines was provided (1.2 [1.0-1.3], and finally, vi) when a vaccination point of contact-defined as an HCW who could provide reliable information on vaccination-was nominated within the nursing home (1.7 [1.3-2.2]). CONCLUSIONS: Urgent and innovative actions are required to increase coverage in HCW. Vaccination programmes should include free on-site vaccination and education campaigns, and particularly target nursing assistants. The results of this nationwide study provide keys for improving influenza vaccination coverage in HCW. Programmes should ensure that information on influenza vaccines is provided by a vaccination point of contact in NH using attractive media. Combining the different prevention measures proposed could increase coverage in NH nationwide by over 50%.

Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display.

This protocol describes the workflow to isolate and engineer fluorescence-activating proteins by yeast surface display. Fluorescence-activating proteins are an emerging class of fluorescent chemogenetic reporters for monitoring gene expression and protein localization in living cells and organisms. They become fluorescent upon binding exogenously applied fluorogenic organic dyes. Efficient fluorescence-activating proteins can be selected from yeast-displayed libraries by iterative rounds of fluorescence-activated cell sorting. The overall strategy is described, as well as a strategy for characterizing the affinity and spectroscopic properties of the selected clones.

Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway.

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

Arylopeptoid oligomers functionalised by combinatorial or sequential on-resin click chemistry using a copper(I)-N-heterocyclic carbene catalyst.

An efficient on-resin click chemistry protocol using a stable copper(I)-N-heterocyclic carbene catalyst is developed for post-functionalization of N-alkylated aminomethylbenzamide oligomers (arylopeptoids). The accessibility to a panel of polyfunctionalized N-substituted aromatic oligoamides by solid-phase synthesis is demonstrated using combinatorial and sequential approaches.

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.