Design of Artificial Transcriptional Activators with Rigid Poly-<scp>l</scp>-proline Linkers

Arora, Paramjit S.; Ansari, Aseem Z.; Best, Timothy P.; Ptashne, Mark; Dervan, Peter B.

doi:10.1021/ja0208355

Cited by 107 publications

(67 citation statements)

References 14 publications

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“…The VP2 minimal AD is a highly acidic 16 amino acid sequence (MLGDFDLDMLGDFDLD) derived from the herpes simplex virus C terminus transactivation domain of VP16 (27). This artificial transactivator is very potent in vitro when linked to a DNA-binding domain (28). We linked this amino acid sequence to PNA78/TAT at its 5′ domain to yield VP2/PNA78/TAT but saw little effect on the γ-globin/luciferase reporter in transfected K562 cells beyond that seen with PNA78/ TAT, likely due to the limits of using the already highly active γ-globin promoter as our test.…”

Section: Detection Of In Vivo Target Binding Of Pna78 In K562 Cells Umentioning

confidence: 99%

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNAbinding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.γ-globin gene reactivation | primary human CD34+ cells

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Section: Detection Of In Vivo Target Binding Of Pna78 In K562 Cells Umentioning

confidence: 99%

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Polyamides have potent biological properties, ranging from selective targeting of viral DNA (5-7), derepression of developmental and disease-causing genes (8,9), and inhibition of tumor growth in vivo (10)(11)(12), to rational design of synthetic transcription factors (13)(14)(15)(16)(17)(18). Remarkably, rationally designed polyamides fed to Drosophila larvae induced classic homeotic patterns of developmental reprogramming (19).…”

mentioning

confidence: 99%

Synthetic genome readers target clustered binding sites across diverse chromatin states

Erwin

Grieshop

Bhimsaria

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to “read” specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

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“…4 and 5). It is important that the distance between the individual pMHC entities in the complex is large, i.e., enforced by polyproline spacers, which in aqueous medium assume a rigid proline II helix of defined length (20,41). For dimeric pMHC class II complexes, the ability to trigger CD4 ϩ T cells decreased also with increasing length of the connecting spacer (up to 90 Å) (42); however, this decrease was much less dramatic than that with pMHC class I dimers (20).…”

Section: Discussionmentioning

confidence: 99%

Soluble MHC-Peptide Complexes Containing Long Rigid Linkers Abolish CTL-Mediated Cytotoxicity

Angelov

Guillaume

Cebecauer

et al. 2006

The Journal of Immunology

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Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and coengage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (≥80 Å) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long Db-gp33 complexes strongly and specifically inhibited the Db-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

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Design of Artificial Transcriptional Activators with Rigid Poly-l-proline Linkers

Cited by 107 publications

References 14 publications

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Synthetic genome readers target clustered binding sites across diverse chromatin states

Soluble MHC-Peptide Complexes Containing Long Rigid Linkers Abolish CTL-Mediated Cytotoxicity

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