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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

Surface plasmon resonance (SPR) titration experiments to study the effect of conjugation on DNA binding properties. Binding of non-functionalized Py–Im polyamide (a + e), Cys-Py–Im-polyamide (b + f), epitope-Py–Im-Polyamide conjugate (c + g) to the cognate DNA sequence during 8 to 12 min injections (25 µL·min−1) over a concentration range of 24 pM to 400 nM in HBS-EP + 0.1% DMSO, pH 7.4; (e) The steady-state binding levels of non-functionalized Py–Im polyamide were fitted individually to a one-site binding model yielding an overall Kd of 1.6 ± 0.1 nM for the non-functionalized Py–Im polyamide binding to the cognate DNA sequence and a Kd of 314 ± 160 nM for the 3 bp mismatch sequence; (f,g) The steady–state binding levels of Cys-Py–Im-polyamide and the epitope-Py–Im polyamide conjugate to the cognate DNA sequence resulted in an overall Kd of 9.3 ± 1.6 and 17.4 ± 0.5 nM, respectively; (d) Response of pre-incubated mixtures containing 100 nM epitope-Py–Im-polyamide conjugate (black line) and anti-HIV antibody binding in a concentration range of 0 to 200 nM (grey lines) to the cognate DNA sequence; (h) Binding level as a function of antibody concentration for the cognate and scrambled DNA sequences.
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ijms-16-12631-f003: Surface plasmon resonance (SPR) titration experiments to study the effect of conjugation on DNA binding properties. Binding of non-functionalized Py–Im polyamide (a + e), Cys-Py–Im-polyamide (b + f), epitope-Py–Im-Polyamide conjugate (c + g) to the cognate DNA sequence during 8 to 12 min injections (25 µL·min−1) over a concentration range of 24 pM to 400 nM in HBS-EP + 0.1% DMSO, pH 7.4; (e) The steady-state binding levels of non-functionalized Py–Im polyamide were fitted individually to a one-site binding model yielding an overall Kd of 1.6 ± 0.1 nM for the non-functionalized Py–Im polyamide binding to the cognate DNA sequence and a Kd of 314 ± 160 nM for the 3 bp mismatch sequence; (f,g) The steady–state binding levels of Cys-Py–Im-polyamide and the epitope-Py–Im polyamide conjugate to the cognate DNA sequence resulted in an overall Kd of 9.3 ± 1.6 and 17.4 ± 0.5 nM, respectively; (d) Response of pre-incubated mixtures containing 100 nM epitope-Py–Im-polyamide conjugate (black line) and anti-HIV antibody binding in a concentration range of 0 to 200 nM (grey lines) to the cognate DNA sequence; (h) Binding level as a function of antibody concentration for the cognate and scrambled DNA sequences.

Mentions: Previous work showed that peptide conjugation can affect both the affinity and specificity of Py–Im polyamides binding to DNA [23,24,25,52]. To study the influence of peptide and protein conjugation, the DNA binding properties of the Py–Im polyamide conjugates were investigated using surface plasmon resonance (SPR). Commercially available streptavidin-functionalized SPR chips were immobilized with biotin-functionalized oligonucleotides (biotin-ODN) that form a hairpin loop resulting in a piece of double stranded DNA containing the cognate sequence for the Py–Im polyamide of interest [53]. Besides the cognate DNA-sequence, we chose to immobilize a scrambled DNA sequence and a sequence containing three mismatches. Increasing concentrations of the reference Py–Im polyamide were injected and flown over the different channels containing one of the three DNA-sequences. The sensorgram of the matching DNA-sequence showed a low dissociation rate indicating tight binding of the non-functionalized Py–Im polyamide to the DNA. The maximum response level of approximately 40 RUs confirmed that the Py–Im polyamide molecule binds to the DNA in a 1:1 stoichiometry (Figure 3a). Fitting the steady state response levels with a 1:1 binding Hill equation (n = 1, no cooperativity) yielded a Kd of 1.6 ± 0.1 nM for the cognate DNA-sequence, which is comparable to the Kd of 2.0 nM reported previously for this Py–Im polyamide sequence using quantititative footprint titrations [41]. In accordance with previous work, a ~100-fold decreased affinity was observed for a sequence that contained three mismatches compared to the cognate DNA sequence (Figure 3e) [54]. Interestingly, introduction of the cysteine-functionalized PEG3-linker at the N-terminus of the Py–Im polyamide resulted in a sixfold decrease in affinity towards the cognate DNA sequence (Figure 3b,f), yielding an apparent Kd of 9.3 ± 1.6 nM. The decreased affinity observed for Cys-Py–Im-polyamide was at least partially due to an enhanced dissociation rate (Figure 3b).


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)

Surface plasmon resonance (SPR) titration experiments to study the effect of conjugation on DNA binding properties. Binding of non-functionalized Py–Im polyamide (a + e), Cys-Py–Im-polyamide (b + f), epitope-Py–Im-Polyamide conjugate (c + g) to the cognate DNA sequence during 8 to 12 min injections (25 µL·min−1) over a concentration range of 24 pM to 400 nM in HBS-EP + 0.1% DMSO, pH 7.4; (e) The steady-state binding levels of non-functionalized Py–Im polyamide were fitted individually to a one-site binding model yielding an overall Kd of 1.6 ± 0.1 nM for the non-functionalized Py–Im polyamide binding to the cognate DNA sequence and a Kd of 314 ± 160 nM for the 3 bp mismatch sequence; (f,g) The steady–state binding levels of Cys-Py–Im-polyamide and the epitope-Py–Im polyamide conjugate to the cognate DNA sequence resulted in an overall Kd of 9.3 ± 1.6 and 17.4 ± 0.5 nM, respectively; (d) Response of pre-incubated mixtures containing 100 nM epitope-Py–Im-polyamide conjugate (black line) and anti-HIV antibody binding in a concentration range of 0 to 200 nM (grey lines) to the cognate DNA sequence; (h) Binding level as a function of antibody concentration for the cognate and scrambled DNA sequences.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490465&req=5

ijms-16-12631-f003: Surface plasmon resonance (SPR) titration experiments to study the effect of conjugation on DNA binding properties. Binding of non-functionalized Py–Im polyamide (a + e), Cys-Py–Im-polyamide (b + f), epitope-Py–Im-Polyamide conjugate (c + g) to the cognate DNA sequence during 8 to 12 min injections (25 µL·min−1) over a concentration range of 24 pM to 400 nM in HBS-EP + 0.1% DMSO, pH 7.4; (e) The steady-state binding levels of non-functionalized Py–Im polyamide were fitted individually to a one-site binding model yielding an overall Kd of 1.6 ± 0.1 nM for the non-functionalized Py–Im polyamide binding to the cognate DNA sequence and a Kd of 314 ± 160 nM for the 3 bp mismatch sequence; (f,g) The steady–state binding levels of Cys-Py–Im-polyamide and the epitope-Py–Im polyamide conjugate to the cognate DNA sequence resulted in an overall Kd of 9.3 ± 1.6 and 17.4 ± 0.5 nM, respectively; (d) Response of pre-incubated mixtures containing 100 nM epitope-Py–Im-polyamide conjugate (black line) and anti-HIV antibody binding in a concentration range of 0 to 200 nM (grey lines) to the cognate DNA sequence; (h) Binding level as a function of antibody concentration for the cognate and scrambled DNA sequences.
Mentions: Previous work showed that peptide conjugation can affect both the affinity and specificity of Py–Im polyamides binding to DNA [23,24,25,52]. To study the influence of peptide and protein conjugation, the DNA binding properties of the Py–Im polyamide conjugates were investigated using surface plasmon resonance (SPR). Commercially available streptavidin-functionalized SPR chips were immobilized with biotin-functionalized oligonucleotides (biotin-ODN) that form a hairpin loop resulting in a piece of double stranded DNA containing the cognate sequence for the Py–Im polyamide of interest [53]. Besides the cognate DNA-sequence, we chose to immobilize a scrambled DNA sequence and a sequence containing three mismatches. Increasing concentrations of the reference Py–Im polyamide were injected and flown over the different channels containing one of the three DNA-sequences. The sensorgram of the matching DNA-sequence showed a low dissociation rate indicating tight binding of the non-functionalized Py–Im polyamide to the DNA. The maximum response level of approximately 40 RUs confirmed that the Py–Im polyamide molecule binds to the DNA in a 1:1 stoichiometry (Figure 3a). Fitting the steady state response levels with a 1:1 binding Hill equation (n = 1, no cooperativity) yielded a Kd of 1.6 ± 0.1 nM for the cognate DNA-sequence, which is comparable to the Kd of 2.0 nM reported previously for this Py–Im polyamide sequence using quantititative footprint titrations [41]. In accordance with previous work, a ~100-fold decreased affinity was observed for a sequence that contained three mismatches compared to the cognate DNA sequence (Figure 3e) [54]. Interestingly, introduction of the cysteine-functionalized PEG3-linker at the N-terminus of the Py–Im polyamide resulted in a sixfold decrease in affinity towards the cognate DNA sequence (Figure 3b,f), yielding an apparent Kd of 9.3 ± 1.6 nM. The decreased affinity observed for Cys-Py–Im-polyamide was at least partially due to an enhanced dissociation rate (Figure 3b).

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