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1.
Nat Methods ; 17(8): 815-821, 2020 08.
Article in English | MEDLINE | ID: mdl-32719532

ABSTRACT

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.


Subject(s)
Fluorescent Dyes/chemistry , Rhodamines/chemistry , Cell Line, Tumor , Humans , Infrared Rays , Microscopy, Fluorescence/methods , Molecular Structure
2.
Nat Chem Biol ; 17(6): 718-723, 2021 06.
Article in English | MEDLINE | ID: mdl-33795886

ABSTRACT

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.


Subject(s)
Fluorescent Dyes/chemistry , Hydrolases/chemistry , Action Potentials/drug effects , Animals , Bioengineering , Calcium/chemistry , Cells, Cultured , Crystallography, X-Ray , Electrophysiological Phenomena , Fluorescent Dyes/chemical synthesis , Hydrolases/chemical synthesis , Kinetics , Molecular Conformation , Molecular Structure , Neurons/drug effects , Primary Cell Culture , Proteins/chemistry , Rats , Rhodamines
3.
Biochemistry ; 61(16): 1665-1668, 2022 08 16.
Article in English | MEDLINE | ID: mdl-35876726

ABSTRACT

Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N1-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.


Subject(s)
RNA , Reverse Transcription , Alkylation , Inosine , RNA/chemistry
4.
Nat Biotechnol ; 41(5): 631-639, 2023 05.
Article in English | MEDLINE | ID: mdl-36593408

ABSTRACT

Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.


Subject(s)
Neurons , Proteins , Neurons/physiology , Promoter Regions, Genetic , Green Fluorescent Proteins/genetics
5.
Neuron ; 111(10): 1547-1563.e9, 2023 05 17.
Article in English | MEDLINE | ID: mdl-37015225

ABSTRACT

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.


Subject(s)
Angiotensin-Converting Enzyme 2 , Rhodopsin , Mice , Animals , Action Potentials/physiology , Rhodopsin/genetics , Neurons/physiology , Mutation/genetics
6.
Sci Rep ; 11(1): 15259, 2021 07 27.
Article in English | MEDLINE | ID: mdl-34315939

ABSTRACT

Geodiversity refers to the variety of geological and physical elements as well as to geomorphological processes of the earth surface. Heterogeneity of the physical environment has an impact on plant diversity. In recent years, the relations between geodiversity and biodiversity has gained attention in conservation biology, especially in the context of climate change. In this study, we assessed the spatial and temporal change in plant's community structure in a semi-arid region, Sayeret Shaked Long Term Ecosystem Research (LTER) station, Israel. Vegetation surveys were conducted on different hillslopes, either with or without rock covers in order to study the spatial trends of hillslope geodiversity. The surveys were conducted for two consecutive years (2016 and 2017), of which the second year was drier and hotter and therefore permitted to investigate the temporal change of plant's community structure. The results of the spatial trends show that (1) geodiversity increases vegetation biodiversity and promotes perennial plants and those of the temporal change show that (2) the positive effect of geodiversity on plants' community structure and species richness is greater in the drier year than that in a wetter year. The main insight is that in these drylands, hillslopes with higher geodiversity appear to buffer the effect of drier years, and supported a more diverse plant community than lower geodiversity hillslopes.

7.
Environ Pollut ; 289: 117788, 2021 Nov 15.
Article in English | MEDLINE | ID: mdl-34332167

ABSTRACT

Crude oil pollution is a global environmental concern since it persists in the environment longer than most conventional carbon sources. In December 2014, the hyper-arid Evrona Nature Reserve, Israel, experienced large-scale contamination when crude oil spilled. The overarching goal of the study was to investigate the possible changes, caused by an accidental crude oil spill, in the leaf reflectance and biochemical composition of four natural habitat desert shrubs. The specific objectives were (1) to monitor the biochemical properties of dominant shrub species in the polluted and control areas; (2) to study the long-term consequences of the contamination; (3) to provide information that will assist in planning rehabilitation actions; and (4) to explore the feasibility of vegetation indices (VIs), along with the machine learning (ML) technique, for detecting stressed shrubs based on the full spectral range. Four measurement campaigns were conducted in 2018 and 2019. Along with the various stress indicators, field spectral measurements were performed in the range of 350-2500 nm. A regression analysis to examine the relation of leaf reflectance to biochemical contents was carried out, to reveal the relevant wavelengths in which polluted and control plants differ. Vegetation indices applied in previous studies were found to be less sensitive for indirect detection of long-term oil contamination. A novel spectral index, based on indicative spectral bands, named the "normalized blue-green stress index" (NBGSI), was established. The NBGSI distinguished significantly between shrubs located in the polluted and in the control areas. The NBGSI showed a strong linear correlation with pheophytin a. Machine learning classification algorithms obtained high overall prediction accuracy in distinguishing between shrubs located in the oil-polluted and the control sites, indicating internal component differences. The findings of this study demonstrate the efficacy of indirect and non-destructive spectral tools for detecting and monitoring oil pollution stress in shrubs.


Subject(s)
Petroleum Pollution , Petroleum , Carbon , Ecosystem , Petroleum Pollution/analysis , Plants
8.
JACS Au ; 1(5): 690-696, 2021 May 24.
Article in English | MEDLINE | ID: mdl-34056637

ABSTRACT

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways-thereby increasing the brightness and photostability of fluorophores-are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

10.
ACS Cent Sci ; 5(9): 1602-1613, 2019 Sep 25.
Article in English | MEDLINE | ID: mdl-31572787

ABSTRACT

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (K L-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

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