| 1. |
- Füchtbauer, Anders Foller, 1984, et al.
(författare)
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Interbase FRET in RNA: from A to Z
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:19, s. 9990-9997
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Characterization of the thermal and photoinduced reactions of photochromic spiropyrans in aqueous solution
- 2013
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 117:43, s. 13561-71
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Tidskriftsartikel (refereegranskat)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.
(författare)
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DNA-Binding Properties of Amidine-Substituted Spiropyran Photoswitches
- 2014
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Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 20:48, s. 15855-15862
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Tidskriftsartikel (refereegranskat)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.
(författare)
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8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:5
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
- 2021
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 143:14, s. 5413-5424
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Tidskriftsartikel (refereegranskat)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.
(författare)
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A Caged Ret Kinase Inhibitor and its Effect on Motoneuron Development in Zebrafish Embryos
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Interbase-FRET binding assay for pre-microRNAs
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)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.
(författare)
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An All-Photonic Molecule-Based Parity Generator/Checker for Error Detection in Data Transmission
- 2013
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 135:28, s. 10230-10233
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors.
- 2015
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)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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| 11. |
- Ferreira, Raphael, 1990, et al.
(författare)
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Photoswitch kinase inhibitors
- 2014
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Ingår i: Purinergic Signalling. - 1573-9538 .- 1573-9546. ; 10:4, s. 767-768
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 12. |
- Nilsson, Jesper, 1984, et al.
(författare)
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A photoswitchable supramolecular complex with release-and-report capabilities
- 2015
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1364-548X .- 1359-7345. ; 51:5, s. 847-850
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Tidskriftsartikel (refereegranskat)abstract
- A self-assembled supramolecular platform has been designed for reversibly controlling the concentration of a compound in solution, via a photochemical reaction. The system utilizes metal-ligand interactions between a Zn-porphyrin dimer and a pyridine-appended dithienylethene (DTE) photoswitch. In addition to reversible compound release, the spectral properties of the release scaffold provide a fluorescence-based reporting function.
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| 13. |
- Nilsson, Jesper, 1984
(författare)
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All-Photonic Control of Biorelevant Processes using Molecular Photoswitches and Caged Compounds
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of light-controlled molecular tools for in situ regulation of biological activity holds great promise e.g. for resolving dynamic aspects of cellular signal transduction and to overcome inherent pharmaceutic limitations such as poor drug selectivity. In this thesis, photoswitchable molecules from the spiropyran, dithienylethene, and azobenzene families as well as an o-nitrobenzyl-type caged compound were used in a variety of constellations intended for biological use. The presented research ranges from spectroscopic studies on isolated molecular interactions in solution to photoactivated kinase inhibition in live zebrafish.The first part of this thesis focuses on novel approaches for photocontrolled drug release, based on a supramolecular competitive binding methodology. Two host-guest systems were investigated for this purpose; a dithienylehtene – porphyrin dimer, and a spiropyran – cucurbit[7]uril (CB7) system. In the first investigated system, it was foundthat drug release could be reversibly controlled by light. The spectral properties of the release scaffold also provided a fluorescence-based reporting function. In the second system, an unexpected loss of spiropyran bistability was observed upon complexation to CB7. Also, the undesired hydrolysis of the spiropyran photoswitch was completelyhalted when bound to the macrocycle. The spiropyran hydrolysis reaction was further examined in a separate study, also included in this thesis. The second and thirds parts focus on photocontrolled small-molecules introduced to living systems. This includes a study on the cytotoxic properties of a DNA-binding spiropyran in live human cancer cells. It was demonstrated how cell-death could be selectively triggered by photoisomerizing the spiropyran to the merocyanine forminside the cells. Part three focuses on photocontrolled inhibition of the, for cancer highly relevant, REarranged during Transfection (RET) kinase. An azobenzenederivedphotoswitch and an o-benzyl-type caged inhibitor based on the pyrazolopyrimidine scaffold were developed and evaluated. Both compounds exhibited light-controlled RET inhibition in an isolated enzyme- and live cell assay.The caged compound was also used to photonically activate inhibition of RET in live zebrafish, confirming uptake and decaging of the compound inside the live organism.
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| 14. |
- Nilsson, Jesper, 1984, et al.
(författare)
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Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers’ RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.
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| 15. |
- Nilsson, Jesper, 1984, et al.
(författare)
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Light-induced cytotoxicity of a photochromic spiropyran
- 2011
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1364-548X .- 1359-7345. ; 47:39, s. 11020-11022
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Tidskriftsartikel (refereegranskat)abstract
- In this work we present a novel water soluble spiropyran photoswitch that can be photonically activated inside live cells from a form that has no significant effect on the cellular survival to a form that induces a dramatic toxic response.
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| 16. |
- Nilsson, Jesper, 1984, et al.
(författare)
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Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA
- 2023
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Ingår i: Physical Chemistry Chemical Physics. - 1463-9084 .- 1463-9076. ; 25:30, s. 20218-20224
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of & sigma;(2PA)(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.
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| 17. |
- Nilsson, Jesper, 1984, et al.
(författare)
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Switching Properties of a Spiropyran-Cucurbit [7] uril Supramolecular Assembly: Usefulness of the Anchor Approach
- 2012
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Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 13:16, s. 3691-3699
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Tidskriftsartikel (refereegranskat)abstract
- A nitrospiropyran, which was modified with a cadaverine-derived anchor, was investigated with respect to its thermally induced isomerizations, hydrolytic stability of the merocyanine form, and photochromic ring closure. The hostguest complexation of the anchor by the cucurbit[7]uril macrocycle, evidenced by absorption titration, NMR spectroscopy, and electrospray ionization mass spectrometry, produced significant improvements of the switching properties of the photochrome: 1) appearance of the merocyanine form about 70 times faster, 2) practically unlimited hydrolytic stability of the merocyanine (two and a half days without any measureable decay), and 3) fast, clean, and fatigue-resistant photoinduced ring closure back to the spiro form. The importance of an adequate molecular design of the anchor was demonstrated by including control experiments with spiropyrans with a shorter linker or without such structural asset.
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| 18. |
- Saarnio, Ville K., et al.
(författare)
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Development of functionalized SYBR green II related cyanine dyes for viral RNA detection
- 2020
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Ingår i: Dyes and Pigments. - : Elsevier BV. - 0143-7208 .- 1873-3743. ; 177
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demand larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.
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| 19. |
- Wypijewska Del Nogal, Anna Wiktoria, 1982, et al.
(författare)
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Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:14, s. 7640-7652
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Tidskriftsartikel (refereegranskat)abstract
- With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Forster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qA(nitro) and tC(nitro), and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.
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