| 1. |
- Füchtbauer, Anders Foller, 1984, et al.
(author)
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Interbase FRET in RNA: from A to Z
- 2019
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:19, s. 9990-9997
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Journal article (peer-reviewed)abstract
- Interbase FRET can reveal highly detailed information about distance, orientation and dynamics in nucleic acids, complementing the existing structure and dynamics techniques. We here report the first RNA base analogue FRET pair, consisting of the donor tC(O) and the non-emissive acceptor tC(nitro). The acceptor ribonucleoside is here synthesised and incorporated into RNA for the first time. This FRET pair accurately reports the average structure of A-form RNA, and its utility for probing RNA structural changes is demonstrated by monitoring the transition from A- to Z-form RNA. Finally, the measured FRET data were compared with theoretical FRET patterns obtained from two previously reported Z-RNA PDB structures, to shed new light on this elusive RNA conformation.
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| 2. |
- Hammarson, Martin, 1984, et al.
(author)
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Characterization of the thermal and photoinduced reactions of photochromic spiropyrans in aqueous solution
- 2013
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 117:43, s. 13561-71
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Journal article (peer-reviewed)abstract
- Six water-soluble spiropyran derivatives have been characterized with respect to the thermal and photoinduced reactions over a broad pH-interval. A comprehensive kinetic model was formulated including the spiro- and the merocyanine isomers, the respective protonated forms, and the hydrolysis products. The experimental studies on the hydrolysis reaction mechanism were supplemented by calculations using quantum mechanical (QM) models employing density functional theory. The results show that (1) the substitution pattern dramatically influences the pKa-values of the protonated forms as well as the rates of the thermal isomerization reactions, (2) water is the nucleophile in the hydrolysis reaction around neutral pH, (3) the phenolate oxygen of the merocyanine form plays a key role in the hydrolysis reaction. Hence, the nonprotonated merocyanine isomer is susceptible to hydrolysis, whereas the corresponding protonated form is stable toward hydrolytic degradation.
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| 3. |
- Hammarson, Martin, 1984, et al.
(author)
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DNA-Binding Properties of Amidine-Substituted Spiropyran Photoswitches
- 2014
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In: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 20:48, s. 15855-15862
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Journal article (peer-reviewed)abstract
- Two amidine-substituted spiropyran derivatives have been characterized with respect to the DNA-binding properties over a broad pH interval. The two derivatives differ in the number of positive charges. By varying the pH, the protonation state of the derivatives is also changed, allowing for additional variations in the charge distribution. We show that the closed spiro isomer does not bind for either of the two derivatives, whereas the open merocyanine forms bind both in the protonated and in the nonprotonated state, but with dramatically different binding constants. Flow-oriented linear dichroism (LD) measurements also show that there are differences in the binding modes between the various forms. We rationalize these differences in terms of structure and charge distribution.
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| 4. |
- Pettersson, Mariell, 1984, et al.
(author)
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8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction
- 2015
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:5
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Journal article (peer-reviewed)abstract
- Small molecule nonpeptidic mimics of alpha-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as alpha-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an alpha-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.
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| 5. |
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| 6. |
- Baladi, Tom, 1991, et al.
(author)
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Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
- 2021
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 143:14, s. 5413-5424
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Journal article (peer-reviewed)abstract
- Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.
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| 7. |
- Bliman, David, et al.
(author)
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A Caged Ret Kinase Inhibitor and its Effect on Motoneuron Development in Zebrafish Embryos
- 2015
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Journal article (peer-reviewed)abstract
- Proto-oncogene tyrosine-protein kinase receptor RET is implicated in the development and maintenance of neurons of the central and peripheral nervous systems. Attaching activity-compromising photocleavable groups (caging) to inhibitors could allow for external spatiotemporally controlled inhibition using light, potentially providing novel information on how these kinase receptors are involved in cellular processes. Here, caged RET inhibitors were obtained from 3-substituted pyrazolopyrimidine-based compounds by attaching photolabile groups to the exocyclic amino function. The most promising compound displayed excellent inhibitory effect in cell-free, as well as live-cell assays upon decaging. Furthermore, inhibition could be efficiently activated with light in vivo in zebrafish embryos and was shown to effect motoneuron development.
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| 8. |
- Bood, Mattias, et al.
(author)
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Interbase-FRET binding assay for pre-microRNAs
- 2021
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Journal article (peer-reviewed)abstract
- The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Forster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA-target binding studies.
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| 9. |
- Bälter, Magnus, 1986, et al.
(author)
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An All-Photonic Molecule-Based Parity Generator/Checker for Error Detection in Data Transmission
- 2013
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 135:28, s. 10230-10233
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Journal article (peer-reviewed)abstract
- The function of a parity generator/checker, which is an essential operation for detecting errors in data transmission, has been realized with multiphotochromic switches by taking advantage of a neuron-like fluorescence response and reversible light-induced transformations between the implicated isomers.
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| 10. |
- Ferreira, Ruben, 1982, et al.
(author)
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Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors.
- 2015
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Journal article (peer-reviewed)abstract
- REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.
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