Interaction study of a lysozyme-binding aptamer with mono- and divalent cations by ACE.
Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na+, K+, Cs+ or divalent (Mg2+, Ca2+, Ba2+) cations and a 30-mer lysozyme-binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30 mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced.
Girardot
Marie
M
Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences, Ecole Nationale Supérieure de Chimie de Paris (Chimie ParisTech), Paris, France.
Gareil
Pierre
P
Varenne
Anne
A
D052157:Aptamers, Nucleotide / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D019075:Electrophoresis, Capillary; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration
D052157:Aptamers, Nucleotide; D002021:Buffers; D002413:Cations, Divalent; D002414:Cations, Monovalent; D009113:Muramidase