Conditionally Activated (“Caged”) Oligonucleotides

Conditionally activated (“caged”) oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical.

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Faculty Opinions recommendation of Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718235138.793512049 ◽

2015 ◽

Author(s):

Sarah Teichmann ◽

Valentina Proserpio

Keyword(s):

Single Cells ◽

Live Tissue ◽

In Vivo Analysis

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Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer’s Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [68Ga]Ga-TC3-OGDOTA

Contrast Media & Molecular Imaging ◽

10.1155/2019/6403274 ◽

2019 ◽

Vol 2019 ◽

pp. 1-17 ◽

Cited By ~ 4

Author(s):

Valeriy G. Ostapchenko ◽

Jonatan Snir ◽

Mojmir Suchy ◽

Jue Fan ◽

M. Rebecca Cobb ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Mouse Models ◽

Caspase 3 ◽

Single Cells ◽

Glucose Deprivation ◽

Apoptotic Cells ◽

The Brain

Apoptosis is a feature of stroke and Alzheimer’s disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [68Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [68Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and β-amyloid oligomers. In vivo, PET showed accumulation of [68Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [68Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [68Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [68Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases.

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Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

Nature Methods ◽

10.1038/nmeth.2804 ◽

2014 ◽

Vol 11 (2) ◽

pp. 190-196 ◽

Cited By ~ 163

Author(s):

Ditte Lovatt ◽

Brittani K Ruble ◽

Jaehee Lee ◽

Hannah Dueck ◽

Tae Kyung Kim ◽

...

Keyword(s):

Single Cells ◽

Live Tissue ◽

In Vivo Analysis

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Protein S-palmitoylation in cellular differentiation

Biochemical Society Transactions ◽

10.1042/bst20160236 ◽

2017 ◽

Vol 45 (1) ◽

pp. 275-285 ◽

Cited By ~ 13

Author(s):

Mingzi M. Zhang ◽

Howard C. Hang

Keyword(s):

Posttranslational Modification ◽

Protein Function ◽

Cellular Differentiation ◽

Protein S ◽

Biological Processes ◽

Global Studies ◽

Technological Advances ◽

Protein Palmitoylation ◽

Spatiotemporal Control

Reversible protein S-palmitoylation confers spatiotemporal control of protein function by modulating protein stability, trafficking and activity, as well as protein–protein and membrane–protein associations. Enabled by technological advances, global studies revealed S-palmitoylation to be an important and pervasive posttranslational modification in eukaryotes with the potential to coordinate diverse biological processes as cells transition from one state to another. Here, we review the strategies and tools to analyze in vivo protein palmitoylation and interrogate the functions of the enzymes that put on and take off palmitate from proteins. We also highlight palmitoyl proteins and palmitoylation-related enzymes that are associated with cellular differentiation and/or tissue development in yeasts, protozoa, mammals, plants and other model eukaryotes.

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Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1342 ◽

2015 ◽

Vol 26 (5) ◽

pp. 843-858 ◽

Cited By ~ 11

Author(s):

Lea Arnold ◽

Sebastian Höckner ◽

Wolfgang Seufert

Keyword(s):

Nuclear Localization ◽

Ubiquitin Ligase ◽

Budding Yeast ◽

Cellular Mechanism ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

In Vivo Analysis ◽

Spatiotemporal Control

In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.

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Inheritance of Chromatin Proteins in Budding Yeast: metabolic gene regulators TUP1, FPR4 and Rpd3L are retained in the mother cell

10.1101/644138 ◽

2019 ◽

Author(s):

Pauline Vasseur ◽

Saphia Tonazzini ◽

Francesc Rubert Castro ◽

Iva Sučec ◽

Arame Fall ◽

...

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Single Cells ◽

Cycle Duration ◽

Imaging Method ◽

Asymmetric Divisions ◽

Phenotypic Transformation ◽

In Vivo Analysis ◽

Chromatin Proteins

AbstractAsymmetric division is a prerequisite for cellular differentiation. Phenotypic transformation during differentiation is a poorly understood epigenetic phenomenon, in which chromatin theoretically plays a role. The assumption that chromatin components segregate asymmetrically in asymmetric divisions has however not been systematically tested. We have developed a live cell imaging method to measure how 18 chromatin proteins are inherited in asymmetric divisions of budding yeast. We show that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3, Fpr4 and Tup1, which are retained in the mother. Mother retention seems to be the norm for low abundance proteins with the exception of Sir2 and the linker histone H1. Our in vivo analysis of chromatin protein behavior in single cells highlights general trends in protein biology during the cell cycle such as coupled protein synthesis and decay, and a correlation between half-lives and cell cycle duration.

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Fluorescent potentiometric dyes for membrane-potential imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168566 ◽

1994 ◽

Vol 52 ◽

pp. 166-167

Author(s):

Leslie M. Loew

Keyword(s):

Membrane Potential ◽

Single Cells ◽

Quantitative Imaging ◽

Optical Recording ◽

Biological Processes ◽

Major Focus ◽

The Past ◽

Excitable Systems ◽

Major Application ◽

The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

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Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

Essays in Biochemistry ◽

10.1042/ebc20190080 ◽

2020 ◽

Vol 64 (2) ◽

pp. 251-261

Author(s):

Jessica E. Fellmeth ◽

Kim S. McKim

Keyword(s):

Chromosome Segregation ◽

New Technologies ◽

Early Prophase ◽

Unique Role ◽

Kinetochore Assembly ◽

M Phase ◽

In Vivo Analysis ◽

Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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