Caged Phosphoproteins

Rothman, Deborah; Petersson, E. James; Vázquez, M. Eugenio; Brandt, G.; Dougherty, Dennis A.; Imperiali, Barbara

doi:10.1021/ja043875c

Cited by 59 publications

(50 citation statements)

References 20 publications

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“…8) In Figure 1, we outlined the desired events for the incorporation of UAA and the undesired possibility of incorporation of natural amino acids at the suppression site. THG73 was previously shown to be orthogonal to the translational machinery in vitro and in vivo when using less mRNA and/or tRNA along with incubations <2 d (Cload et al 1996;Saks et al 1996;England et al 1999;Shafer et al 2004;Rothman et al 2005). We have shown that with increasing all three of the aforementioned conditions, producing so-called excessive conditions, THG73 can be aminoacylated in vivo.…”

Section: Lysrs Does Not Aminoacylate Thg73 Tqas9 or Tqasmentioning

confidence: 90%

“…Unlike its mathematical counterpart, there can be degrees of orthogonality when referring to tRNAs. While the orthogonality of THG73 has been evaluated using in vitro translation in Escherichia coli (Cload et al 1996), wheat germ (England et al 1999), and rabbit reticulocyte lysate (Rothman et al 2005), the primary application of THG73 has been for in vivo translation in Xenopus oocytes, which is the focus of the present work. The orthogonality of THG73 is acceptable when injecting low quantities of mutant mRNA and/or tRNA-UAA with incubation times <2 d (Saks et al 1996;Shafer et al 2004).…”

Section: Introductionmentioning

confidence: 99%

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Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Rodriguez¹,

Lester²,

Dougherty³

2007

RNA

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The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.

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Section: Lysrs Does Not Aminoacylate Thg73 Tqas9 or Tqasmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Rodriguez¹,

Lester²,

Dougherty³

2007

RNA

Self Cite

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“…Using solid-phase peptide synthesis, a caged tyrosine was incorporated into a small inhibitory peptide 15 . Other peptides have been caged using the same approach 16 , 52 , 53 , 54 .…”

Section: Caging Peptides and Proteinsmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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“…Since caging of ATP was fi rst reported in 1978 by Kaplan et al, several different photolabile groups have been introduced to turn biomolecules temporarily inactive (1 -3) . Examples of caged biomolecules are neurotransmitters (4) , nucleotides (4) , peptides (5,6) , siRNA (7) or DNA (8) . The most widely used caged neurotransmitter so far is glutamate for which different protecting groups have been applied (9) .…”

Section: Application Of Light In Nanomedicine Light For Triggered Relmentioning

confidence: 99%

“…Another approach of light sensitive nanoparticles currently being investigated uses nano-impellers. Nano-impellers are nanomechanical systems allowing the spatiotemporal drug release upon illumination, turning them into an attractive application for clinical trials (5,6,18,19) . A clear disadvantage of many published systems is the requirement for light energy in the UV range, limiting their application due to phototoxicity and the very limited penetration range of short wavelength light in biological tissues.…”

Section: Application Of Light In Nanomedicine Light For Triggered Relmentioning

confidence: 99%

Why not just switch on the light?: light and its versatile applications in the field of nanomedicine

Lehner¹,

Hunziker²

2016

Nano Online

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Over the last decade, the emerging fi eld of nanomedicine has undergone rapid progresses. Different internal and external stimuli like pH, temperature, radiation, ultrasound or light have been introduced to expand the diagnostic and therapeutic options of various applications within the fi eld. This review focuses on the novel application of light in the fi eld of nanomedicine as a mechanism to control drug delivery, release and biochemical and genetic functionality at the target. The fi eld of functional nanomaterials for medicine, and in particular of light responsive nanocarriers, polymers and biomolecules offer new therapeutic options but also requires substantial further research to render this approach broadly applicable in clinical practice.

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Caged Phosphoproteins

Cited by 59 publications

References 20 publications

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Why not just switch on the light?: light and its versatile applications in the field of nanomedicine

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