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Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

Janssen BM, van Ommeren SP, Merkx M - Int J Mol Sci (2015)

Bottom Line: The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR).Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates.The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands. b.m.g.janssen@tue.nl.

ABSTRACT
The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

No MeSH data available.


Related in: MedlinePlus

(a) Chemical structure and schematic representation of the reference Py–Im polyamide; (b) RP-HPLC and m/z-spectra of the purified acetylated Py–Im polyamide; (c) Chemical structure and schematic representation of the Py–Im polyamide sequence containing the PEG3-linker and t-BuSH-protected cysteine (Cys-Py–Im-polyamide) after cleavage from the resin using Dp; (d) RP-HPLC and m/z-spectra of the purified Cys-Py–Im-polyamide. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
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ijms-16-12631-f001: (a) Chemical structure and schematic representation of the reference Py–Im polyamide; (b) RP-HPLC and m/z-spectra of the purified acetylated Py–Im polyamide; (c) Chemical structure and schematic representation of the Py–Im polyamide sequence containing the PEG3-linker and t-BuSH-protected cysteine (Cys-Py–Im-polyamide) after cleavage from the resin using Dp; (d) RP-HPLC and m/z-spectra of the purified Cys-Py–Im-polyamide. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.

Mentions: Figure 1 shows the chemical structure and electro spray ionization mass spectrometry (ESI-MS) characterization of the final product, Cys-Py–Im-polyamide, as well as the Py–Im polyamide reference compound with an N-terminal acetyl group. Both compounds were cleaved via aminolysis of the ester on the resin using 3-(dimethylamino)-propylamine (Dp). The positively charged tertiary amine that is formed at the C-termini of the Py–Im polyamides provides an additional electrostatic interaction between the Py–Im polyamide and DNA. Typical overall yields after reversed phase high-performance liquid chromatography (RP-HPLC) were 20% for the Py–Im polyamide reference and 14% for Cys-Py–Im-polyamide, which is comparable to yields reported previously for Py–Im polyamides of similar sequences [40].


Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation.

Janssen BM, van Ommeren SP, Merkx M - Int J Mol Sci (2015)

(a) Chemical structure and schematic representation of the reference Py–Im polyamide; (b) RP-HPLC and m/z-spectra of the purified acetylated Py–Im polyamide; (c) Chemical structure and schematic representation of the Py–Im polyamide sequence containing the PEG3-linker and t-BuSH-protected cysteine (Cys-Py–Im-polyamide) after cleavage from the resin using Dp; (d) RP-HPLC and m/z-spectra of the purified Cys-Py–Im-polyamide. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490465&req=5

ijms-16-12631-f001: (a) Chemical structure and schematic representation of the reference Py–Im polyamide; (b) RP-HPLC and m/z-spectra of the purified acetylated Py–Im polyamide; (c) Chemical structure and schematic representation of the Py–Im polyamide sequence containing the PEG3-linker and t-BuSH-protected cysteine (Cys-Py–Im-polyamide) after cleavage from the resin using Dp; (d) RP-HPLC and m/z-spectra of the purified Cys-Py–Im-polyamide. Mwexp was obtained after deconvolution of the m/z-spectrum. Mwcalc is the calculated average molecular weight.
Mentions: Figure 1 shows the chemical structure and electro spray ionization mass spectrometry (ESI-MS) characterization of the final product, Cys-Py–Im-polyamide, as well as the Py–Im polyamide reference compound with an N-terminal acetyl group. Both compounds were cleaved via aminolysis of the ester on the resin using 3-(dimethylamino)-propylamine (Dp). The positively charged tertiary amine that is formed at the C-termini of the Py–Im polyamides provides an additional electrostatic interaction between the Py–Im polyamide and DNA. Typical overall yields after reversed phase high-performance liquid chromatography (RP-HPLC) were 20% for the Py–Im polyamide reference and 14% for Cys-Py–Im-polyamide, which is comparable to yields reported previously for Py–Im polyamides of similar sequences [40].

Bottom Line: The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR).Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates.The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands. b.m.g.janssen@tue.nl.

ABSTRACT
The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py-Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py-Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py-Im polyamides. The effect of Py-Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py-Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py-Im-polyamide conjugates. The practical use of protein-Py-Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

No MeSH data available.


Related in: MedlinePlus