Determination of the Equilibrium Constant and Rate Constant of Protein-Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis.
Anal Chem
; 91(13): 8532-8539, 2019 07 02.
Article
em En
| MEDLINE
| ID: mdl-31136154
ABSTRACT
Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant ( Kd) and rate constant ( koff) of protein-oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein-oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of Kd and koff. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine Kd and koff for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding Kd and koff of protein-oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
/
Eletroforese Capilar
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Proteínas de Fluorescência Verde
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Aptâmeros de Nucleotídeos
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Proteínas MutS
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Canadá