Dataset Information

A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

ABSTRACT: Nitrate (NO₃ ^-) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an attractive strategy. To monitor the flux of nitrate, we developed a non-invasive genetically encoded fluorescence resonance energy transfer (FRET)-based tool named "FLIP-NT" that monitors the real-time uptake of nitrate in the living cells. The developed nanosensor is suitable for real-time monitoring of nitrate flux in living cells at subcellular compartments with high spatio-temporal resolution. The developed FLIP-NT nanosensor was not affected by the pH change and have specificity for nitrate with an affinity constant (K _d) of ?5 ?M. A series of affinity mutants have also been generated to expand the physiological detection range of the sensor protein with varying K _d values. It has been found that this sensor successfully detects the dynamics of nitrate fluctuations in bacteria and yeast, without the disruption of cellular organization. This FLIP-NT nanosensor could be a very important tool that will help us to advance the understanding of nitrate signaling.

SUBMITTER: Fatima U

PROVIDER: S-EPMC7689916 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

Fatima Urooj U Ameen Fuad F Soleja Neha N Khan Parvez P Almansob Abobakr A Ahmad Altaf A

ACS omega 20201112 46

Nitrate (NO<sub>3</sub> <sup>-</sup>) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an ...[more]

PMID: 33251465

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

Publications

A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

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