Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein

Abstract

AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

NMR monitoring of the SELEX process to confirm enrichment of structured RNA

“…HTS data were compared with the sequences of the 36 clones in the 8R RNA pool that were determined using the capillary sequencing method 45 . The fractions of each aptamer were determined as follows: S1, 27.8%; S2, 22.2%; S3, 16.7%.…”

“…We focused on the aptamers S1 and S2, which were sufficiently abundant to observe them in the pool, although other aptamers that showed higher affinity to RD than these two aptamers were obtained in a previous study 45 . Given that HTS data revealed that the fraction of S1 and S2 was high at 5–8R, we prepared isolated S1 and S2 aptamers and compared the imino proton spectra of 5–8R RNA pools with those of S1 and S2 (Fig.…”

“…As a control experiment, SELEX was performed as described previously 45 except that the selection step was omitted, a procedure referred to as “Neutral SELEX” 15 .…”

“…RNA pools and aptamers (S1 and S2) were prepared as described previously 45 . The poly (A-U) RNA pool was prepared using a DNA template (5′-CGGAAAAAAAAAAAAAAAAAAAAG-40N-ATTTTTTTTTTTTTTTTTTTTCCC TATAGTGAGTCGTATTA -3′; the T7 promoter sequence is underlined, and 40N represents 40 nucleotides (nt) of random sequence and primers (Fwd1; 5′-TAATACGACTCACTATAGGGAAAAAAAAAAAAAAAAAAAAT-3′ and Rev1; 5′-CGGAAAAAAAAAAAAAAAAAAAAG-3′) (Hokkaido System Science Co., Ltd, Sapporo, Japan).…”

“…In a previous study, we obtained high-affinity RNA aptamers against the AML1 Runt domain (RD) after 8 rounds of SELEX 45 . To evaluate NMR monitoring for the enrichment of RNA aptamers during the SELEX process, we measured 1D imino proton spectra of the RNA pools obtained in our previous study.…”