PNA‐Encoded Synthesis (PES) of a 10 000‐Member Hetero‐Glycoconjugate Library and Microarray Analysis of Diverse Lectins

Novoa, Alexandre; Machida, Takuya; Barluenga, Sofía; Imberty, Anne; Winssinger, Nicolas

doi:10.1002/cbic.201402280

Cited by 36 publications

(26 citation statements)

References 62 publications

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“…The N‐termini of PNAs were capped with either Cy3 or biotin for quantification of individual sequences within the library through hybridization onto a DNA microarray containing the complementary sequences. The PNA sequences were designed to minimize cross‐hybridization while having a balanced G/C content and have been used to tag multiple libraries of small molecules . Both libraries were prepared with a d ‐Arg residue at the beginning and the end of the PNA sequence to ensure good solubility of the library.…”

Section: Resultsmentioning

confidence: 99%

Caprin‐1 Promotes Cellular Uptake of Nucleic Acids with Backbone and Sequence Discrimination

Galli

Sadhu

Masi

et al. 2019

Helvetica Chimica Acta

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The cellular delivery of oligonucleotides has been a major obstacle in the development of therapeutic antisense agents. PNAs (Peptide Nucleic Acid) are unique in providing a modular peptidic backbone that is amenable to structural and charge modulation. While cationic PNAs have been shown to be taken up by cells more efficiently than neutral PNAs, the generality of uptake across different nucleobase sequences has never been tested. Herein, we quantified the relative uptake of PNAs across a library of 10 000 sequences for two different PNA backbones (cationic and neutral) and identified sequences with high uptake and low uptake. We used the high uptake sequence as a bait for target identification, leading to the discovery that a protein, caprin-1, binds to PNA with backbone and sequence discrimination. We further showed that purified caprin-1 added to cell cultures enhanced the cellular uptake of PNA as well as DNA and RNA. Figure 2. Single sequence specificity uptake of Ser-PNAs in HeLa cells. A) Maximum projection of Z-stacks images comparing the internalization of Cy3 labelled HU sequence (high uptake sequence) and LU sequence (low uptake sequence) and Arg/Ser peptides. Cell membrane was stained with CellMask Green Plasma Membrane Stain (Thermo Fisher Scientific, Waltham, MA, USA). B) Plots of Cy3 fluorescent signal quantification in cells. Number of cells analyzed: Ser-HU n = 61, Ser-LU n = 68, Arg-HU n = 70, Arg-LU n = 57, Ser-peptide n = 19, Arg-peptide n = 14 (bars are averages � SEM, unpaired t-test, p-values < 0.0001). C) HU and LU nucleobase sequences. The modified residues (either α-arginine modification or γ-serine modification) are shown in red. Scale bar: 10 μm.

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Section: Resultsmentioning

confidence: 99%

Caprin‐1 Promotes Cellular Uptake of Nucleic Acids with Backbone and Sequence Discrimination

Galli

Sadhu

Masi

et al. 2019

Helvetica Chimica Acta

Self Cite

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“…1). 36,[44][45][46][47][48][49][50] We reasoned that this method could allow us to discriminate between covalent and non-covalent ligands using a denaturing wash that would denature proteins and compromise noncovalent ligand-protein interactions. However, aside from peptide-based libraries targeting proteases, 33,[37][38] no DNA or PNA-encoded library specifically designed to engage diverse protein targets in covalent interactions had been reported.…”

Section: Resultsmentioning

confidence: 99%

Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moieties

et al. 2016

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DNA-encoded chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2

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“…royalsocietypublishing.org/journal/rsfs Interface Focus 9: 20180080 glycan interactions in a biological context [30,31], allowing for controlled assembly of glycan microheterogeneity [32] and the selection of optimal glycoconjugate-receptor pairs using dynamic assembly [19] and directed evolution [33,34] of glycoconjugates.…”

Section: Nanoscale Mimetics Of Macromolecular Constituents Of the Glymentioning

confidence: 99%

Synthetic glycoscapes: addressing the structural and functional complexity of the glycocalyx

Purcell

Godula

2019

Interface Focus.

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One contribution of 12 to a theme issue 'Synthetic glycobiology'.The glycocalyx is an information-dense network of biomacromolecules extensively modified through glycosylation that populates the cellular boundary. The glycocalyx regulates biological events ranging from cellular protection and adhesion to signalling and differentiation. Owing to the characteristically weak interactions between individual glycans and their protein binding partners, multivalency of glycan presentation is required for the high-avidity interactions needed to trigger cellular responses. As such, biological recognition at the glycocalyx interface is determined by both the structure of glycans that are present as well as their spatial distribution. While genetic and biochemical approaches have proven powerful in controlling glycan composition, modulating the three-dimensional complexity of the cell-surface 'glycoscape' at the sub-micrometre scale remains a considerable challenge in the field. This focused review highlights recent advances in glycocalyx engineering using synthetic nanoscale glycomaterials, which allows for controlled de novo assembly of complexity with precision not accessible with traditional molecular biology tools. We discuss several exciting new studies in the field that demonstrate the power of precision glycocalyx editing in living cells in revealing and controlling the complex mechanisms by which the glycocalyx regulates biological processes.

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PNA‐Encoded Synthesis (PES) of a 10 000‐Member Hetero‐Glycoconjugate Library and Microarray Analysis of Diverse Lectins

Cited by 36 publications

References 62 publications

Caprin‐1 Promotes Cellular Uptake of Nucleic Acids with Backbone and Sequence Discrimination

Caprin‐1 Promotes Cellular Uptake of Nucleic Acids with Backbone and Sequence Discrimination

Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moieties

Synthetic glycoscapes: addressing the structural and functional complexity of the glycocalyx

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