| 1. |
- Barnoin, Guillaume, et al.
(författare)
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Intermolecular dark resonance energy transfer (DRET): Upgrading fluorogenic DNA sensing
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:12
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Demirbay, Baris
(författare)
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Concepts and biomedical applications of excitation-modulated transient state monitoring of fluorescence emitters
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fluorescence methods have developed very strongly in the last decade, allowing single- molecule detection sensitivity, high specificity, high time- and spatial resolution, as well as high readout speeds. Together, this makes fluorescencea very central readout modality for biomolecular and cellular studies. The photophysics of the fluorescence emitters, fluorophores, used is of central importance or the performance. Here, the photostability and brightness ofthe fluorophores, but also their blinking properties set the limits of the performance. Fluorophore blinking, arising from photo-induced, non-fluorescent transient states of fluorophores, can however also be taken advantage of cellular and biomolecular studies. Blinking is a prerequisite for essentially allso-called fluorescence-based super-resolution techniques. Moreover, blinkingis often sensitive to micro-environmental parameters such as pH, oxygen concentrations,redox conditions and viscosity. This follows from the fact that the underlying, non-luminescent, dark transient states typically have 103 to 106 longer lifetimes than the fluorescent excited states of the fluorophores, thereby giving fluorescent molecules in the dark states more time to interactwith their surrounding in biological environment. It can thus be utilized as an alternative readout parameter to provide useful information on molecules and cells and their environments, beyond what can be monitored by traditional fluorescence methods. This thesis takes as one starting point the transient state (TRAST) spectroscopy technique, designed to monitor such long-lived, dark transient states, including triplet, photo-oxidation, photo-reduction and photo-isomerized states of fluorophores, by measuring how the time-averaged fluorescence signal detected in the sample is changed upon systematically varying the excitation modulation.The major focus of the present thesis work is to further extent the use of long-lived dark transient states of fluorescence emitters in solution, lipid membranes and live cells. For this different TRAST modalities were adapted and developed, and then demonstrated as useful characterization methods. First, we showed how the relaxed brightness requirements of TRAST made it possible to characterize the photo-physical properties of the high triplet yield carboxy-fluorescein dye and its brominated derivatives (paper I). By widefield TRAST measurements, we demonstrated its capability to sense heavy atom effect of bromine and iodide atoms, and how they affected the triplet and long-lived photo-oxidation states and their transitions rates. Next, we developed and demonstrated a concept based on TRAST method and its ability to distinguish fluorophores with different blinking properties as a way to perform fluorescence-based barcoding and multiplexing. This concept, demonstrated by exploiting the by TRAST well distinguishable photophysical transitions of two fluorescent dyes, which emit in the same spectral range, was demonstrated in paper II. In the same work, we also developed a TRAST modality for microfluidic measurements of molecules and lipid vesicles, on which the bar-coding concept could be demonstrated on-the-fly, as the molecules and vesicles passed through the microfluidic channel. Furthermore, with TRAST implemented in camera-based wide-field microscopy, multicolor barcoded images of cells with high spatial resolution could be further investigated for the first time due to the specific blinking dynamics of these labels. The last two papers of this thesis describe further extensions of the TRAST concept, and the monitoring of fluorescence blinking to live cell studies. In paper III, TRAST in a widefield microscopy setting was employed in combination with FCS to study the folding of dye-labelled RNA strands into G-quadruplex structures in solution and live cells using photo-isomerization kinetics of cyanine dye as a readout parameter. Here, we took advantage of the high sensitivity of cyanine dye photoisomerization, to viscosity and steric constraints,and the resulting blinking of the cyanines, to monitor conformation changes of RNAs in live cells. Finally, in paper IV, we demonstrated how it by TRAST imaging, taking advantage of the photo-induced dark states of a mitochondrial localization fluorophore (n-Nonyl Acridine Orange, NAO), is possible to give this localization probe environmental sensing properties as well.To sum up, the experimental findings and papers included in this thesis show that fluorescence blinking represent a rich source of information for biomolecular and cellular studies. By the TRAST technique, and the variants further adapted and developed in this work, it is shown that it possible tocapture this rich source of complementary information in a broad range of samples. The work in this thesis suggest that further combination of classical fluorescence readouts and a continued development of different TRAST modalities will open yet new windows and provide insights into molecular interaction studies in biological research.
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| 3. |
- Enander, Karin, 1972-, et al.
(författare)
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A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte
- 2008
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:9, s. 1864-1870
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Tidskriftsartikel (refereegranskat)abstract
- We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore (IN*/IT*) upon binding of Fab57P. When the peptide was labeled in the C terminus, the IN*/I T* ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats. © 2008 American Chemical Society.
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| 4. |
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| 5. |
- Jiang, Kai, 1988, et al.
(författare)
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Annealing of ssDNA and compaction of dsDNA by the HIV-1 nucleocapsid and Gag proteins visualized using nanofluidic channels
- 2019
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 52, s. e2-e2
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Tidskriftsartikel (refereegranskat)abstract
- The nucleocapsid protein NC is a crucial component in the human immunodeficiency virus type 1 life cycle. It functions both in its processed mature form and as part of the polyprotein Gag that plays a key role in the formation of new viruses. NC can protect nucleic acids (NAs) from degradation by compacting them to a dense coil. Moreover, through its NA chaperone activity, NC can also promote the most stable conformation of NAs. Here, we explore the balance between these activities for NC and Gag by confining DNA-protein complexes in nanochannels. The chaperone activity is visualized as concatemerization and circularization of long DNA via annealing of short single-stranded DNA overhangs. The first ten amino acids of NC are important for the chaperone activity that is almost completely absent for Gag. Gag condenses DNA more efficiently than mature NC, suggesting that additional residues of Gag are involved. Importantly, this is the first single DNA molecule study of full-length Gag and we reveal important differences to the truncated Δ-p6 Gag that has been used before. In addition, the study also highlights how nanochannels can be used to study reactions on ends of long single DNA molecules, which is not trivial with competing single DNA molecule techniques.
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| 6. |
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| 7. |
- Jiang, Kai, 1988, et al.
(författare)
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The HIV-1 nucleocapsid chaperone protein forms locally compacted globules on long double-stranded DNA
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:8, s. 4550-4563
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Tidskriftsartikel (refereegranskat)abstract
- The nucleocapsid (NC) protein plays key roles in Human Immunodeficiency Virus 1 (HIV-1) replication, notably by condensing and protecting the viral RNA genome and by chaperoning its reverse transcription into double-stranded DNA (dsDNA). Recent findings suggest that integration of viral dsDNA into the host genome, and hence productive infection, is linked to a small subpopulation of viral complexes where reverse transcription was completed within the intact capsid. Therefore, the synthesized dsDNA has to be tightly compacted, most likely by NC, to prevent breaking of the capsid in these complexes. To investigate NC's ability to compact viral dsDNA, we here characterize the compaction of single dsDNA molecules under unsaturated NC binding conditions using nanofluidic channels. Compaction is shown to result from accumulation of NC at one or few compaction sites, which leads to small dsDNA condensates. NC preferentially initiates compaction at flexible regions along the dsDNA, such as AT-rich regions and DNA ends. Upon further NC binding, these condensates develop into a globular state containing the whole dsDNA molecule. These findings support NC's role in viral dsDNA compaction within the mature HIV-1 capsid and suggest a possible scenario for the gradual dsDNA decondensation upon capsid uncoating and NC loss.
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