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Modulation of Aptamer–Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study
Authors:Alexey V. Samokhvalov  Irina V. Safenkova  Sergei A. Eremin  Artem N. Bonchuk  Oksana G. Maksimenko  Nikolai N. Sluchanko  Anatoly V. Zherdev  Boris B. Dzantiev
Affiliation:1.A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow 119071, Russia; (A.V.S.); (I.V.S.); (S.A.E.); (N.N.S.); (A.V.Z.);2.Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia;3.Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia; (A.N.B.); (O.G.M.)
Abstract:Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer–ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer–ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs’ features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5′-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3′) and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer–ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer–ssDNA complex. The ssDNAs’ having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer–OTA interactions. The ssDNAs’ having less than 18 hydrogen bonds did not affect the aptamer–OTA affinity. The location of ssDNA’s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer–ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA’s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn’t affecting the G-qaudruplex’s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.
Keywords:aptamers, G-quadruplex, DNA–  DNA interactions, isothermal microcalorimetry, fluorescence anisotropy, circular dichroism, ochratoxin A
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