| 1. |
- Becker, Juliane, et al.
(författare)
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MULTIPLE ORTHOGONAL LABELLING OF OLGONUCLEOTIDES
- 2016
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Patent (populärvet., debatt m.m.)abstract
- In Summary, the present invention concerns a method for multiple orthogonal labelling of oligonucleotides, preferably RNA or DNA, by simultaneously performing the inverse Diels-Alder reaction (DAinv) and the copper-catalyzed click reaction (CuAAC), wherein the method is employed in a single step by just adding the different reaction components together and incubating the aqueous reaction mixture prefer ably for one hour at room temperature. In detail, the reaction components are one or more N-modified labels, a copper compound, a stabilizing ligand, a reducing agent and one or more electron-deficient label-modified dienes that are added together with an at least double-modified oligonucleotide having one more nucleotides containing one or more N3-re active groups and one or more electron-rich dienophiles, wherein a terminal alkyne moiety is preferably used as N3-re active group(s) and afrans-cyclooctene moiety or norbornene is preferably used as electron-rich dienophile(s), more pref erably frans-cyclooctene. Therefore, the present invention provides a one-pot method for post-synthetic multiple orthogonal labeling of oligonucleotides, which allows the site-specific introduction of more than one label, preferably of at least two labels into oligonucleotides after solid-phase synthesis, wherein the DAinv takes place on the dienophile modification only and the CuAAC selectively takes place on the N3-reactive group modification.
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| 2. |
- Manz, Christoph, et al.
(författare)
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Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using Single-Molecule FRET Spectroscopy
- 2018
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495.
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Konferensbidrag (refereegranskat)abstract
- Binding of the ligand S-adenosyl-L-methionine (SAM) produces major structural changes in the SAM-I riboswitch (RS) and thereby regulates gene expression via transcription termination. As yet, the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I RS have not been deeply investigated. We have studied its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy. smFRET histograms of differently FRET-labeled constructs were so complicated that they could only be resolved with the help of kinetic experiments on immobilized riboswitches and hidden Markov modeling (HMM) analysis. At least four conformational states were identified, both in the presence and the absence of SAM. We determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds were found to coexist under all conditions; SAM binding induced only a gradual increase in the population of terminator states. Conformational transitions were much faster with bound SAM, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.
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| 3. |
- Manz, C., et al.
(författare)
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Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch
- 2017
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 13, s. 1172-1178
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Tidskriftsartikel (refereegranskat)abstract
- S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.
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| 4. |
- Manz, C., et al.
(författare)
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The multi-state energy landscape of the SAM-I riboswitch: : A single-molecule Förster resonance energy transfer spectroscopy study
- 2018
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 148:12
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Tidskriftsartikel (refereegranskat)abstract
- RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.
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| 5. |
- Meyer, Sascha W, et al.
(författare)
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APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP
- 2011
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Ingår i: European Molecular Biology Organization. - : Wiley. - 1460-2075. ; 30, s. 2266-2280
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Tidskriftsartikel (refereegranskat)abstract
- Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock‐in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα‐DM) by crossing APPsα‐KI mice onto an APLP2‐deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα‐DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα‐DM muscle showed fragmented post‐synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα‐DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABAA receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.
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| 6. |
- Samanta, Ayan, et al.
(författare)
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A modified dinucleotide for site-specific RNA-labelling by transcription priming and click chemistry
- 2014
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Ingår i: Chemical Communications. - 1359-7345 .- 1364-548X. ; :11
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Tidskriftsartikel (refereegranskat)abstract
- An improved strategy for RNA labelling using an alkyne-carrying dinucleotide is reported. This involves near-quantitative priming by phage RNA-polymerases followed by conjugation of different labels using click chemistry. Moreover, these transcripts bear a ligation compatible 5′-end, and thus through ligation the terminal label can be transformed to an internal one.
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| 7. |
- Schoch, Juliane, et al.
(författare)
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Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry
- 2012
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 23:7, s. 1382-1386
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Tidskriftsartikel (refereegranskat)abstract
- Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide–alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels–Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M–1 s–1). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.
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| 8. |
- Winz, Marie-Luise, et al.
(författare)
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Functionalization of RNA oligonucleotides
- 2013
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Patent (populärvet., debatt m.m.)abstract
- The inventors of the present invention developed a two-step method for the post-synthetic enzymatic incorporation of chemical modifications or rather functionalization of RNA oligonucleotides for their labelling. In a first step, the RNA oligonuc - leotide is tailed by the incorporation of a convertible azido-modified (N -modified) nucleotide at the 3'-terminus in an enzymatic re00 action with a nucleotidyl transferase, e.g., the enzyme poly(A) polymerase (PAP) and an N3-modified nucleoside triphosphate (N3 - modified NTP). In a second step, the functionalization is accomplished with an azide-reactive group, e.g., via a copper-catalyzed © 1,3-dipolar cycloaddition reaction between the incorporated N 3 -modified nucleotide and a functional group containing a terminal alkyne moiety (copper-catalyzed click reaction), via a strain-promoted [3 + 2] dipolar cycloaddition between the incorporated N -modified nucleotide and a functional group containing a cyclooctyne moiety (strain-promoted click reaction), or via an azide-phosphine conjugation between the incorporated N -modified nucleotide and a functional group containing a phosphine moiety (Staudinger ligation). To achieve internal chemical modifications or rather functionalization of RNA oligonucleotides, an additional step can be in - troduced between the first and the second step, in which a second RNA oligonucleotide is enzymatically attached to the N -modified nucleotide at the 3'-terminus of the first step, thus converting the 3'-terminal N3-modified nucleotide into an internal N3-modified nucleotide that can be further converted in one of the aforementioned reactions.
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| 9. |
- Winz, Marie-Luise, et al.
(författare)
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Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:10, s. e78-
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Tidskriftsartikel (refereegranskat)abstract
- The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 30-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strainpromoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 20-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 20-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.
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