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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

Binding of protein-Py–Im-polyamide conjugate to a surface modified with the cognate DNA sequence. (a) Response of ECFP-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM to the cognate DNA sequence; (b) Response of CNA35-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM binding to the cognate DNA sequence. Experiments were conducted in HBS-EP + 0.1% DMSO, pH 7.4 and using 10 min injections (25 µL·min−1).
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ijms-16-12631-f004: Binding of protein-Py–Im-polyamide conjugate to a surface modified with the cognate DNA sequence. (a) Response of ECFP-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM to the cognate DNA sequence; (b) Response of CNA35-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM binding to the cognate DNA sequence. Experiments were conducted in HBS-EP + 0.1% DMSO, pH 7.4 and using 10 min injections (25 µL·min−1).

Mentions: Finally, the SPR binding assay was used to assess the DNA-binding properties of the protein-Py–Im-polyamide conjugates (Figure 4). Although DNA binding was clearly observed, both the ECFP-Py–Im-polyamide and the CNA35-Py–Im-polyamide conjugate showed complex association kinetics. At relatively high concentrations, the initial rapid increase in response was followed by an unexpected drop in response levels until a steady-state level was reached. A possible explanation for this complex binding behavior could be competitive binding by a compound with a higher affinity and lower molecular weight. Care was taken to remove the excess of non-conjugated Cys-Py–Im-polyamide following NCL, either be repeated concentration/dilution cycles using centrifugal filters (for ECFP and EYFP) or using Ni-affinity chromatography (for CNA35). Nonetheless, we cannot exclude the possibility that some of the Cys-Py–Im-polyamide sticks to the protein due to its strongly hydrophobic nature. Although the steady-state levels are higher for the protein-Py–Im-polyamide conjugates compared to the smaller Cys-Py–Im-polyamide or epitope-Py–Im-polyamide conjugate, it is also clear that the binding is not saturated even at the highest concentration of protein-Py–Im-polyamide tested (400 nM), which indicates that the DNA affinity is further attenuated for the protein-Py–Im-polyamide conjugates compared to the Cys-Py–Im and epitope-Py–Im-polyamide conjugates.


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)

Binding of protein-Py–Im-polyamide conjugate to a surface modified with the cognate DNA sequence. (a) Response of ECFP-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM to the cognate DNA sequence; (b) Response of CNA35-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM binding to the cognate DNA sequence. Experiments were conducted in HBS-EP + 0.1% DMSO, pH 7.4 and using 10 min injections (25 µL·min−1).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4490465&req=5

ijms-16-12631-f004: Binding of protein-Py–Im-polyamide conjugate to a surface modified with the cognate DNA sequence. (a) Response of ECFP-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM to the cognate DNA sequence; (b) Response of CNA35-Py–Im-polyamide conjugate binding in a concentration range of 1.56 to 400 nM binding to the cognate DNA sequence. Experiments were conducted in HBS-EP + 0.1% DMSO, pH 7.4 and using 10 min injections (25 µL·min−1).
Mentions: Finally, the SPR binding assay was used to assess the DNA-binding properties of the protein-Py–Im-polyamide conjugates (Figure 4). Although DNA binding was clearly observed, both the ECFP-Py–Im-polyamide and the CNA35-Py–Im-polyamide conjugate showed complex association kinetics. At relatively high concentrations, the initial rapid increase in response was followed by an unexpected drop in response levels until a steady-state level was reached. A possible explanation for this complex binding behavior could be competitive binding by a compound with a higher affinity and lower molecular weight. Care was taken to remove the excess of non-conjugated Cys-Py–Im-polyamide following NCL, either be repeated concentration/dilution cycles using centrifugal filters (for ECFP and EYFP) or using Ni-affinity chromatography (for CNA35). Nonetheless, we cannot exclude the possibility that some of the Cys-Py–Im-polyamide sticks to the protein due to its strongly hydrophobic nature. Although the steady-state levels are higher for the protein-Py–Im-polyamide conjugates compared to the smaller Cys-Py–Im-polyamide or epitope-Py–Im-polyamide conjugate, it is also clear that the binding is not saturated even at the highest concentration of protein-Py–Im-polyamide tested (400 nM), which indicates that the DNA affinity is further attenuated for the protein-Py–Im-polyamide conjugates compared to the Cys-Py–Im and epitope-Py–Im-polyamide conjugates.

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