Design of Photocaged Puromycin for Nascent Polypeptide Release and Spatiotemporal Monitoring of Translation

Buhr, Florian; Kohl-Landgraf, Jörg; Dieck, Susanne tom; Hanus, Cyril; Chatterjee, Deep; Hegelein, Andreas; Schuman, Erin M.; Wachtveitl, Josef; Schwalbe, Harald

doi:10.1002/ange.201410940

Cited by 42 publications

(6 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, the band of the carbonyl stretch mode of the carbamate linker (1722 cm –1 ) decreases on the same time scale as the formation of CO 2 . The release of CO 2 by uncaging a product linked via a carbamate has been shown in the past and monitors in this case the uncaging of glutamic acid. , Accordingly, we assign the absorption change observed in the stationary UV/vis measurements (Figure A) to uncaging of glutamate indicated by the isosbestic point and determined an uncaging quantum yield of 1.5% (IR and UV/vis measurements on the reference compound II+III reveal an uncaging quantum yield, which is 1 order of magnitude lower than that of I+II+III ; for further information, see the SI).…”

mentioning

confidence: 53%

Sensitized Two-Photon Activation of Coumarin Photocages

Hammer

Falahati

Jakob

et al. 2018

J. Phys. Chem. Lett.

Self Cite

View full text Add to dashboard Cite

Here we report the design of a new coumarin-based photolabile protecting group with enhanced two-photon absorption. Two-photon excited fluorescence (TPEF), color-tuned ultrafast transient absorption spectroscopy and infrared (IR) measurements are employed to photochemically characterize the newly designed ATTO 390-DEACM-cargo triad. Increased two-photon cross-section values of the novel cage in comparison to the widely used protecting group DEACM ([7-(diethylamino)coumarin-4-yl]methyl) are extracted from TPEF experiments. Femtosecond pump-probe experiments reveal a fast intramolecular charge transfer, a finding that is confirmed by quantum chemical calculations. Uncaging of glutamate is monitored in IR measurements by photodecarboxylation of the carbamate linker between the photolabile protecting group and the glutamate, showing the full functionality of the novel two-photon activatable photocage.

show abstract

mentioning

confidence: 53%

Sensitized Two-Photon Activation of Coumarin Photocages

Hammer

Falahati

Jakob

et al. 2018

J. Phys. Chem. Lett.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Unlike radioactive labeling, small molecule drugs used in animal studies possess no harm to experimentalists, and the collected tissues could be used for transcriptome sequencing, and proteomic and metabolomic studies. It also expands the existing puromycin-based technique for spatiotemporal monitoring of nascent peptide release (19).…”

Section: Discussionmentioning

confidence: 99%

Organ-specific translation elongation rates measured by in vivo ribosome profiling

Gerashchenko

Péterfi

Gladyshev

2018

Preprint

View full text Add to dashboard Cite

Protein synthesis and degradation are intricate biological processes involving more than a hundred proteins operating in a highly orchestrated fashion. Despite the progress, few options are available to access translation in live animals as the increase in animal's complexity limits the repertoire of experimental tools that could be applied to observe and manipulate processes within animal's body, organs, and individual cells. It this study, we developed a labeling-free method for measuring organ-and cell-type specific translation elongation rates. It is based on a time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ among mouse organs and determined them to be 6.8, 5.2, and 4.4 amino acids per sec for liver, kidney, and skeletal muscle, respectively. protein synthesis | translation | elongation | ribosome profiling | mouse P rotein synthesis is regulated both at the transcriptional (availability of mRNA) and translational (the number of active ribosomes, translational efficiency, the speed of elongation and the frequency of initiation events) levels. This complexity presents endless possibilities for malfunctioning. In turn, the vast majority of cellular functions are carried by proteins, making any aberrations in their biosynthesis potentially harmful. Modern in vitro methods allow unsurpassed precision and flexibility, e.g. real-time translation dynamics of a single molecule can be monitored with fluorescent microscopy (1, 2), and transcriptome-wide snapshots of translation with a single nucleotide resolution can be achieved by means of ribosome profiling (3-6). In contrast to research performed in cells, few options are currently available to investigate translation in live animals. It is particularly challenging to assay specific steps of translation, i.e. elongation, initiation or termination rate, in vivo. Several clever approaches were designed, including animals fed with amino acid supplements labeled with a stable isotope (7) or radioactively labeled amino acids. To estimate the average translation elongation speed, short-term pulses of radioactively labeled amino acids can be injected directly into the portal vein of an animal (8). The radiation accumulation rate can be used to project the average translation elongation rate (8,9); however, tissue samples are rendered by this method unsuitable for many other molecular biology assays.We were guided by these challenges and the need to assess various steps of translation in various organs of live animals at an individual gene level. In this work, we present a technique to directly assess translation in vivo and measure organ-and cell-specific translation elongation rates and other features of protein synthesis. Our method requires no radioactive labeling or transgenic reporters and can be applied to a variety of small animals such as a mouse or a rat. It...

show abstract

“…The extinction coefficient ( ϵ max ) value was taken from the literature (6210 m −1 cm −1 for NVOC at 365 nm, 4100 m −1 cm −1 for DMNPB at 346 nm, and 7500 m −1 cm −1 for HANBP at 397 nm), the absorbance value ( A ) at the relevant λ max value was determined from the UV/Vis spectra of the films, and the pathlength ( l ) corresponded to the swollen thickness of hydrogel film (≈0.007 cm) and was obtained for each film from fluorescence correlation spectroscopy . The binding efficiency was calculated from the ratio between the absorbance of peptide solutions before ( A i ) and after ( A a ) coupling to hydrogel, according to the following equation:

true binding efficiency [%] = \frac{A_{i} - A_{a}}{A_{i}} \times 100

…”

Section: Methodsmentioning

confidence: 99%

Photoactivatable Adhesive Ligands for Light‐Guided Neuronal Growth

et al. 2018

View full text Add to dashboard Cite

Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.

show abstract

Design of Photocaged Puromycin for Nascent Polypeptide Release and Spatiotemporal Monitoring of Translation

Cited by 42 publications

References 35 publications

Sensitized Two-Photon Activation of Coumarin Photocages

Sensitized Two-Photon Activation of Coumarin Photocages

Organ-specific translation elongation rates measured by in vivo ribosome profiling

Photoactivatable Adhesive Ligands for Light‐Guided Neuronal Growth

Contact Info

Product

Resources

About