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Kinetic and thermodynamic analyses of interaction between a high-affinity RNA aptamer and its target protein Ryo Amano, Kenta Takada, Yoichiro Tanaka, Yoshikazu Nakamura, Gota Kawai, Tomoko Kozu, and Taiichi Sakamoto Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00748 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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Biochemistry

Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and its Target Protein Ryo Amano,† Kenta Takada,† Yoichiro Tanaka,‡ Yoshikazu Nakamura,§,ǁ Gota Kawai,† Tomoko Kozu,# and Taiichi Sakamoto*,†

†

Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of

Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan ‡

Facility for RI Research and Education, Instrumental Analysis Center, Yokohama National

University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan §

Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo,

Shirokanedai, Minato-ku, Tokyo 108-8639, Japan ǁ

Ribomic Inc., 3-16-13 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan

#

Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama 362-0806, Japan

1 ACS Paragon Plus Environment

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Keywords SELEX, RNA aptamer, kinetics, thermodynamics


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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 SELEX. 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. 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, 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 on the biophysics of aptamer binding should be useful for


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understanding the mechanism of RNA–protein interaction and optimizing and modifying RNA aptamers.


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Aptamers are short single-stranded nucleic acid molecules that are selected in vitro from large random sequence libraries based on their high affinity for target molecules by a process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX).1–5 The target molecules range from small molecules, such as nucleotides, cofactors, and amino acids, followed by peptides, polysaccharides, and proteins, to complex structures, such as whole cells, viruses, and single-celled organisms. Similar to antibodies, aptamers show high affinity and specificity against target molecules as well as additional advantages, including their small size and ease of chemical production.6–8 Thus, aptamers are promising therapeutic agents. However, the details of the biophysical aspects of high-affinity binding of aptamers to target molecules are still unknown. The transcription factor AML1, also known as RUNX1, is one of the key regulators of hematopoiesis and is involved in regulating the transcription of a range of blood cell-specific genes.9–11 The region evolutionarily most conserved in the AML1 protein is the DNA-binding domain, referred to as the Runt domain (RD), which is responsible for binding to a specific DNA sequence.12,13 The tertiary structure of RD has been investigated by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.13–16 These studies revealed that RD recognizes


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the DNA consensus sequence “YGYGGTY” (Y = pyrimidine) using two loop-containing regions and a C-terminal tail, which clamp around the sugar–phosphate backbone. The C-terminal tail and one of the loop regions interact with the major groove. Three guanines of the consensus sequence are recognized by three arginine residues that form hydrogen bonds in the major groove. The other loop region interacts with the minor groove. Complex formation is further supported by several interactions between the side chains and backbone of RD and the sugar– phosphate backbone of DNA. In addition to its role in the regulation of normal hematopoiesis, the AML1 gene is a protooncogene. One of the most common chromosomal translocations in acute myeloid leukemia, t(8;21), generates the oncogenic fusion gene AML1-ETO, which is generally considered to act as a transcriptional repressor by interacting with other proteins.17–29 RNA aptamers that bind to RD were previously studied regarding their potential utility in the diagnosis and treatment of AML1related diseases.30–32 We have already obtained an RNA aptamer (Apt1-S) that shows higher affinity (Kd = 0.99 ± 0.02 nM) than the Runt-binding double-stranded DNA element (RDE, Kd = 9.6 ± 0.2 nM).31,32


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Nonspecific RNA binding to proteins is assumed to be due mainly to electrostatic interactions between positively charged proteins and the negatively charged phosphate backbone of RNA. Therefore, in this study, to remove the nonspecific binding of RNAs to RD, we performed SELEX under a high salt concentration and isolated RNA aptamers targeting RD with a higher affinity than previously reported aptamers. To reveal the kinetic and thermodynamic aspects of the high affinity of the selected aptamer, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) measurements were performed. Our data show that the aptamer binds to a larger surface area of RD than that of RDE.

EXPERIMENTAL PROCEDURES Protein expression and purification. The coding sequence of an AML1 N-terminal fragment (amino acids 51–188, referred to as the Runt domain) was cloned into the pQE80 vector (QIAGEN). Recombinant hexahistidine-tagged AML1–RD was expressed in Escherichia coli strain BL21 (DE3) (Novagen). The crude extract from a 1-L culture was applied to a 5-mL His trap HP column (GE Healthcare) equilibrated with buffer A [20 mM Tris–HCl (pH 8.0), 500 mM


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NaCl, and 1 mM DTT] and 5 mM imidazole. A Protease Inhibitor Cocktail Set, EDTA-Free (Merck), was added to E. coli lysate. After washing with buffer A and 5 mM imidazole, the protein was eluted using a 5–500 mM imidazole gradient, followed by dialysis against buffer B [20 mM sodium phosphate (pH 6.5), 50 mM NaCl, and 1 mM DTT]. The sample was then applied to a 5-mL Hi trap SP column (GE Healthcare) equilibrated with buffer B and eluted using a 0–1 M NaCl gradient. The purified protein was then dialyzed against buffer C [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 300 mM potassium acetate, 50% glycerol, and 1 mM DTT] and stored at −25°C. The concentration of RD was determined based on its molecular absorption coefficient at 280 nm. SELEX.

A

DNA

template

[5′-CTCTCATGTCGGCCGTTA-40N-

CGTCCATTGTGTCCCTATAGTGATCGTATTA-3′; T7 promoter sequence is underlined, and 40N

represents

40

nucleotides

(nt)

of

random

TAATACGACTCACTATAGGGACACAATGGACG-3′),

sequence], and

primer

primer

1 2

(5′(5′-

CTCTCATGTCGGCCGTTA-3′) were synthesized and purchased from Hokkaido System Science Co., Ltd. An initial DNA library was constructed from 400 pmol synthetic DNA


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template. Thus, a pool of, in theory, 2.4 × 1014 different RNA molecules randomized over 40 nucleotides was prepared by in vitro transcription using T7 RNA polymerase. Before the selection process, the purified RNAs were heat denatured at 95°C for 5 min, refolded by snap cooling to 4°C, and incubated in binding buffer [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 500 mM potassium acetate, 5% glycerol, 0.05% Triton-X100, and 5 mM βmercaptoethanol] for 10 min at 37°C. To reduce undesired, nonspecific adsorption of RNAs to RD or resins, 2–5 mg/mL tRNA (Invitrogen) was added to the folded RNA pool. Two types of resins, Ni-agarose (QIAGEN) and Talon metal affinity (Clontech) resins, were alternatively used for the affinity capture of the recombinant hexahistidine-tagged RD. Negative selection was performed before and after positive selection. For negative selection, the folded RNA pools were transferred to 3 µL of Ni agarose and Talon metal affinity resins without protein immobilization, followed by incubation for 30 min at room temperature. Then, the supernatant containing unbound RNAs was collected and subjected to positive selection. For positive selection, the RNA pools were transferred to 3 µL of the indicated resin immobilized with RD, followed by incubation for 30 min at room temperature. After the samples had been washed for the indicated


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times with 400 µL of binding buffer, tightly bound RNAs were eluted from the resin via the addition of 200 µL of binding buffer containing 500 mM imidazole, subjected to phenol– chloroform extraction, and precipitated using ethanol. The recovered RNAs were reverse transcribed using ReverTra Ace reverse transcriptase (TOYOBO) and amplified by PCR, employing KOD-Plus-DNA polymerase (TOYOBO), with primers 1 and 2, followed by T7 transcription. After eight rounds of SELEX, cDNAs were cloned into the pGEM-T Easy vector (Promega) and sequenced. Details of the conditions for selection in each round are listed in Table S1. Aptamer preparation. S4, S4-S, and Apt1-S were synthesized by in vitro transcription. The template of S4-S was amplified from a cloning vector containing S4 by PCR. PCR products were purified by phenol–chloroform extraction and precipitated using ethanol. The template of Apt1-S was purchased from Hokkaido System Science Co., Ltd. All RNAs were purified by denaturing PAGE and their concentrations were determined based on their molecular absorption coefficients at 260 nm. All RNAs were annealed by heating at 95°C for 5 min, followed by snap cooling on ice.


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Surface plasmon resonance (SPR) assays. SPR assays were performed as previously described using a BIAcore X instrument (GE Healthcare).31 The 5′-biotinylated dT16 oligomer was immobilized to flow cell 2 on the surface of a streptavidin sensor chip (GE Healthcare) at a level of approximately 800 Resonance Units. The sensor chip was washed and equilibrated in SPR buffer [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 0.1% Tween 20, and 1 mM DTT] containing 300 mM or 1 M potassium acetate at 298 K. The 3′-A16-tagged RNAs or 3′-A16-tagged RDE (5′-GTCGTTTGCGGTTTGGGGAAAAAAAAAAAAAAAAA-3′ or 5′TCCCCAAACCGCAAACGAC-3′) dissolved in SPR buffer was immobilized to approximately 100 Resonance Units in flow cell 2 at a flow rate of 90 µL/min. RDE and the 40N random RNA pool were used as positive and negative controls, respectively. RD (0.625–2400 nM) or Rev peptide (62.5–2000 nM) in SPR buffer were injected into flow cells 1–2 of the sensor chip for 90 s and dissociated for 180 s. To regenerate the sensor chip, the bound material was completely removed by injecting 4 M urea. The signal of flow cell 2 was subtracted from that of flow cell 1 to eliminate nonspecific interactions. The sensorgrams were analyzed using BIA evaluation software (GE Healthcare). A Langmuir (1:1) binding model was used to analyze the association


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rate constant kon and the dissociation rate constant koff (M–1 s−1 and s−1, respectively). The dissociation constant Kd was also determined as the ratio of koff and kon as follows: Kd = koff/kon, and is represented by the mean ± standard error of three independent measurements. Isothermal titration calorimetry (ITC) experiments. ITC experiments were performed at 288, 293, 298, or 303 K with a Microcal iTC200 (Malvern). S4-S, RDE (5′GTCGTTTGCGGTTTGGGGA-3′/5′-TCCCCAAACCGCAAACGAC-3′),

and

RD

were

dissolved in ITC buffer [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 300 mM potassium acetate, 10% glycerol, and 1 mM DTT]. S4-S or RDE solution in an injection syringe (100 µM) was injected into RD solutions in a cell (10 µM). The volume of each injection was 2 µL, except that of the first injection was 0.4 µL. All measurements involved 19 injections at 150s intervals, a syringe rotational speed of 1250 rpm, and a reference power of 5.0 µcal s−1. To subtract the heat of dilution of the samples, the oligonucleotide samples were injected into the buffer alone. Each area of the heat pulse was integrated, and the resulting values were plotted as a function of the molar ratio. The stoichiometry of binding N, the dissociation constant Kd, and the enthalpy change ∆H were obtained according to single-site-binding model curve fitting provided


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by the instrument software Origin 7.0 (Microcal), although the c value for ITC was higher than 1000.33-35 The Gibbs free energy change ∆G and the entropy change ∆S were calculated based on Kd and ∆H. Each thermodynamic parameter is represented by the mean ± standard error of three independent measurements. The binding heat capacity change ∆Cp is obtained by measuring the binding enthalpy change as a function of temperature.36 Negative heat capacity change is usually associated with specific recognition and burial of an apolar surface area.

RESULTS Selection and identification of RNA aptamers that bind to RD. We isolated RNA aptamers against RD by SELEX using an initial RNA pool of 73-nt RNA with 40-nt random sequences, referred to as the 40N RNA pool. To select tightly bound aptamers, we performed selection under a high salt concentration and progressively increased the selection stringency by increasing the number of washes and concentration of tRNA as the competitor and decreasing the target concentration for positive selection. Following eight rounds of SELEX, variants of the RNA pool were cloned and sequenced. A total of 36 clones were randomly selected, and 11


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independent sequences, S1–S11, were isolated (Table 1). The binding affinity of these 11 sequences to RD was analyzed by SPR, and the dissociation constants (Kd) of all of these clones were lower than 0.3 nM. The Kd of S1, which is most frequently observed, was 0.27 ± 0.02 nM, and that of S4, which shows the highest binding affinity, was 0.044 ± 0.002 nM. Secondary structure prediction and truncation of S4. The secondary structure of S4, which contains a multibranched loop, was predicted using the vs_subopt program (http://www.rna.it-chiba.ac.jp/~vsfold/vs_subopt/)37,38 (Figure 1A). Based on the predicted secondary structure of S4, single-stranded primer binding sequences were truncated and a stem (5′-GGA/CCGA-3′) was added for effective transcription by T7 RNA polymerase and for stabilization of the terminal stem structure, which resulted in a length of 52 nucleotides (Figure 1B). The SPR analysis showed that the Kd of S4-S was 0.034 ± 0.004 nM, which is approximately 300 and 30 times lower than that of RDE (Kd = 10 ± 1 nM) and Apt1-S (Kd = 1.1 ± 0.1 nM), respectively (Figures 1, 2, and Table 2). The difference in the RD-binding affinities between S4-S, RDE, and Ap1-S is mainly attributed to the lower dissociation rate (koff) of S4S/RD interaction than those of the RDE–RD and Apt1-S–RD interactions.


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To confirm the specificity of S4-S binding to RD, the binding of S4-S, RDE, and Apt1-S to Rev peptide derived from the arginine-rich motif (ARM) of HIV Rev protein as a negative control was examined by SPR (Figure S1 and Table S2). It is well known that the Rev peptide binds to the Rev-response element (RRE) RNA with high affinity (Kd = 6.8 nM at 293 K; ITC experiment) and specificity.39,40 S4-S, RDE, and Apt1-S exhibited low affinity in the micromolar range against Rev peptide because of non-specific binding. It was suggested that S4-S binds to RD with high affinity and specificity. Salt concentration dependence of S4-S binding to RD. The salt concentration dependence of S4-S binding to RD was investigated because S4 was selected under the highly stringent conditions with a high salt concentration. Although the binding affinity of S4-S to RD was decreased as the potassium acetate concentration was increased to 1 M, S4-S retained its binding affinity (Kd = 3.5 ± 0.4 nM) (Table 2 and Figure 2D). On the other hand, the binding of RDE to RD was too weak to be detected using 1 M potassium acetate (Table 2 and Figure 2E). Apt1-S also markedly diminished the binding affinity (Kd = 2800 ± 200 nM) (Table 2 and Figure


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2F). The loss of affinity of S4-S at a high salt concentration was because of a decrease in kon without a change in koff. Thermodynamic analysis of S4-S for RD binding. In addition to kinetic analysis using SPR, the thermodynamics of the interactions between S4-S and RD was analyzed by ITC. An exothermic heat pulse was observed after each injection of S4-S or RDE into RD (Figure 3 and Figure S2). Although the Kd values of S4-S involve error due to inaccuracy of curve fitting (c > 1000),33-35 the thermodynamic parameters obtained at 288, 293, 298, and 303 K are shown in Table 3. The stoichiometry N for RD binding to S4-S, RDE, or Apt1-S is almost 1; the difference may depend on the precision of S4-S, RDE, Apt1-S and/or RD concentrations or the proportion of inactive RD in the solution. Comparison of the Kd values indicates that S4-S retains almost the same affinity to RD in the range of 288−303 K, whereas its affinity to RDE declines with increasing temperature. Furthermore, S4-S binds to RD with an approximately 10-fold higher affinity (Kd = 5 ± 3 nM at 298 K) than its affinity to RDE (Kd = 66 ± 5 nM at 298 K). The observed change in enthalpy (∆H) for S4-S binding (∆H = −56 ± 1 kcal mol−1 at 298 K) is greater than that for RDE binding (∆H = −25.5 ± 0.4 kcal mol−1 at 298 K). However, the change in


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entropy (T∆S) for S4-S binding (−T∆S = −45 ± 1 kcal mol−1 at 298 K) is smaller than that for RDE binding (−T∆S = −15.7 ± 0.4 kcal mol−1 at 298 K). These results indicate that S4-S binding to RD is contributed to by higher enthalpic interaction than its binding to RDE, and its unfavorable T∆S is compensated for by the favorable ∆H. To elucidate the heat capacity change (∆Cp) for RD binding to S4-S and RDE, ∆H values versus temperatures were plotted (Figure 4). The heat capacity change plot revealed that the binding of S4-S or RDE to RD becomes increasingly exothermic with increasing temperature. The ∆Cp values were calculated from the linear fit of ∆H versus temperature data in the 288−303 K range. Both S4-S–RD and RDE–RD interactions exhibit negative heat capacity change in this temperature range, and the ∆Cp value of S4-S–RD interaction (−0.82 ± 0.14 kcal mol−1 K−1) is lower than that of RDE–RD interaction (−0.52 ± 0.05 kcal mol−1 K−1). The T∆S values versus temperatures are plotted for S4-S and RDE, revealing that the binding entropy change for both S4-S and RDE becomes increasingly unfavorable with increasing temperature, akin to the binding enthalpy change (Figure S3).


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DISCUSSION

In this work, we obtained high-affinity RNA aptamers against RD under highly stringent conditions (Table 1). Among the selected aptamers, S4 showed the highest binding affinity (Kd = 0.044 ± 0.002 nM), whereas the Kd of the most frequently observed S1 was 0.27 ± 0.02 nM. It was previously reported that the most frequently observed aptamer showed fast association rather than high affinity.41 However, the association of S1 with RD was slower than that of other aptamers, except for S8, in this case. Thus, the reason why the most frequently observed S1 did not show the highest binding affinity could include bias in the efficiency of transcription or reverse-transcription PCR in the SELEX scheme.42 The sequence of S1 may be suitable for amplification in the SELEX scheme. We succeeded in developing S4-S, which is a shortened variant of S4 and shows higher binding affinity than the parent S4. According to the computational prediction of secondary structure, S4-S comprises a multibranched loop, two hairpin stem-loops, and a distal stem. It was suggested that the truncated 3′- and 5′-regions of S4 are dispensable for RD binding and the stabilization of the distal stem structure is favorable for RD binding.


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It has often been observed that the Kd values obtained by SPR are smaller than those obtained by ITC.43,44 Although the reason for this difference is not clearly understood, it may be caused by the following two differences. First, the solution components are different among the two experiments. The solution used for ITC contained 10% glycerol to prevent RD from precipitation, whereas no glycerol was included in the SPR solution. Thus, glycerol at the interface between S4-S and RD may prevent interaction between them. Second, basic physical conditions are different among the two methods. SPR involves solid-phase measurement of RD binding to S4-S immobilized on a sensor chip, whereas ITC involves solution-phase measurement of RD binding to free S4-S. Therefore, it is assumed that the entropy change upon association of RD with an immobilized S4-S at SPR is smaller than that of RD with a free S4-S in solution at ITC. 45,46 These differences may further lead to a difference in Kd values observed by SPR and ITC. SPR analyses revealed that S4-S binds to RD with faster association and slower dissociation compared with RDE and Apt1-S (Table 2). Although the binding affinity of RDE to RD was completely diminished and that of Apt1-S was extremely reduced when 1 M potassium acetate


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was used, S4-S retained its binding activity under the same conditions. Selection under a high salt concentration would result in retention of the binding activity. Furthermore, kinetic analysis of S4-S binding shows that koff did not change and kon decreased by increasing salt concentration, whereas kinetic analysis of Apt1-S binding shows that koff increased and kon decreased. Ions counteract the electrostatic interactions between the negatively charged phosphate backbone of S4-S and the basic residues in RD; this may lead to a decrease in kon.47–50 The result that koff was not changed could be explained by good geometric complementarity between the interacting surfaces S4-S and RD. In some cases of the aptamer that binds to its target protein, the crystal structure revealed that the aptamer binds to its target protein with good shape complementarity and a wide range of contours on protein surfaces.51–54 Thus, we are attempting a structural study of the complex for S4-S and RD to reveal the complementarity of their shape. Figure 5 shows that the partitioning of the contributions of ∆H and T∆S to S4-S/RD interaction and RDE–RD interaction markedly differs. S4-S binding to RD relies on a large favorable ∆H offset by a strongly unfavorable T∆S, whereas RDE binding to RD displays relatively less favorable ∆H and less unfavorable T∆S. The thermodynamic parameters provide


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information about the interaction between biomolecules.36,40,43–47,55–62 A more favorable ∆H for S4-S–RD interaction suggests more favorable interactions than the RDE–RD interactions; this may be caused by specific binding with good surface complementarity formed by optimal hydrogen bonding and van der Waals interactions. The highly unfavorable T∆S in S4-S binding to RD could arise from decreased solvation entropy and/or decreased conformational entropy in the bound state. Furthermore, the larger negative ∆Cp of S4-S binding than that of RDE binding suggests that a larger apolar surface area is buried upon complex formation for S4-S binding than that for RDE binding (Table 3 and Figure 4). Thus, it was suggested that the unfavorable entropy change is due to conformational restraint in the bound form. These ITC findings are consistent with the fact that the dissociation of S4-S from RD occurs very slowly, as observed by SPR. In summary, SPR and ITC experiments have shown the binding kinetics and thermodynamics of S4-S binding to RD. From the obtained results, it was proposed that in comparison with RDE, S4-S binds to RD with stronger long-range electrostatic force in the early stage of the association and then S4-S changes its conformation and recognizes the larger surface area of RD by optimal hydrogen bonding, van der Waals contact, and/or hydrophobic interaction. Recently, a


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combination of structural, kinetic, and thermodynamic analyses has been used for the effective screening and rational optimization of lead chemical compounds and antibody molecules48,61,62. However, optimization and modification of aptamers have been performed by trial and error. Thus, these biophysical analyses may provide some valuable information for the optimization and modification of aptamers. Furthermore, these approaches should be useful for understanding the mechanism underlying various RNA–protein interactions.


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ASSOCIATED CONTENT Supporting Information

In vitro selection conditions (Table S1), Kinetic parameters of RNA aptamers or RDE binding to Rev peptide (Table S2), SPR analysis of RNA aptamers or RDE binding to Rev peptide (Figure S1), ITC profiles at various temperatures of S4-S/RD interaction and RDE/RD interaction (Figure S2), Temperature dependence of entropy for S4-S binding (Figure S3). These materials are available online free of charge at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-47-478-0317; Fax: +81-47-478-0317;

Email: [email protected]

Funding


Biochemistry

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This study was supported by research grants of Innovative Area, Structural Cell Biology (Number 23121528), and the Strategic Research Foundation Grant-aided Project for Private Universities (Number S1101001) from The Ministry of Education, Sports, Culture, Science and Technology (MEXT) of Japan.

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors thank Prof. Masato Katahira and Prof. Takashi Nagata for helpful discussions. We also thank Ms. Masako Hirose for technical support with ITC measurements.

ABBREVIATIONS SELEX, systematic evolution of ligands by exponential enrichment; AML1, acute myeloid leukemia 1; RD, Runt domain; RDE, Runt-binding double-stranded DNA element; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry


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Biochemistry

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Table 1. Sequences of RD-binding RNA aptamers and their kinetic parameters a Clone ID S1

S2

S3

S4

S5

S6

S7

S8

S9

Sequence (5′ to 3′) b

kon

koff

(×106 M−1s−1)

(×10−3 s−1)

10/36

8.5 ± 0.4

2.3 ± 0.1

0.27 ± 0.02

8/36

9.6 ± 0.2

1.1 ± 0.1

0.11 ± 0.01

6/36

9.7 ± 0.3

2.1 ± 0.1

0.21 ± 0.01

3/36

9.1 ± 0.1

0.40 ± 0.02

0.044 ± 0.002

2/36

9.3 ± 0.4

1.1 ± 0.1

0.11 ± 0.13

2/36

13 ± 2

1.5 ± 0.1

0.12 ± 0.09

1/36

11.6 ± 0.2

0.7 ± 0.3

0.056 ± 0.003

1/36

6.8 ± 0.3

0.53 ± 0.8

0.077 ± 0.004

1/36

13.1 ± 0.3

0.82 ± 0.2

0.063 ± 0.002

Frequency

gggacacaauggacgUGUCGGCCCUGCCGUGUAACGC UGGCGCGGGAUGUUCUCCuaacggccgacaugagag gggacacaauggacgGCCCAGCCACCUAGAGCGAGCG CGCAAUGGAGACCCAUUGuaacggccgacaugagag gggacacaauggacgUGUCGGCCCUGCCGUGUAACGC UGGCGCGGGACUUCUCCuaacggccgacaugagag gggacacaauggacgGCCCUGCCACGAUAGCGGCGCG GGAAGUAAAGUAUACACCuaacggccgacaugagag gggacacaauggacgUGUCGGCCCUGCCGUGUAAUGC UGGCGCGGGACGUUCUCCuaacggccgacaugagag gggacacaauggacgGUCAGCCACCACUGUGCGGCGA GCGGAAGCACACCGUCCGuaacggccgacaugagag gggacacaauggacgGCCCUGCCACCUAGAGCGAGCG CGCAAUGGAGACCCAUUGuaacggccgacaugagag gggacacaauggacgGCCCUGCCACGAAGGCGGCGCG CAGGCUACCCGCACCUGuaacggccgacaugagag gggacacaauggacgAUGCCGGCCCUGCCACACCAAU GCGGCGCGGUCAAUAGACuaacggccgacaugagag


Kd (nM)

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Biochemistry

S10

S11

a

gggacacaauggacgGCCCAGCCACCUAGUGCGAGCG CGCAAUGGAGACCCAUUGuaacggccgacaugagag gggacacaauggacgACGCCGGCCCUGCCACACCAAU GCGGCGCGGUCAAUAGACuaacggccgacaugagag

1/36

10.7 ± 0.5

0.97 ± 0.4

0.090 ± 0.001

1/36

13.7 ± 0.2

1.27 ± 0.03

0.093 ± 0.002

All SPR experiments were performed in 20 mM sodium phosphate (pH 6.5) with 2 mM magnesium acetate, 300 mM

potassium acetate, 0.1% Tween 20, and 1 mM DTT at 298 K. The kon, koff, and Kd values represent the mean ± SE (n = 3).

b

Lower-case letters indicate nucleotides derived from the primer-binding sites.


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Table 2. Kinetics of the binding of S4-S, RDE, or Apt1-S to RD at 0.3 M or 1 M potassium acetate a

0.3 M potassium acetate

1 M potassium acetate

kon

koff

Kd

kon

koff

Kd

(×106 M−1s−1)

(×10−3 s−1)

(nM)

(×106 M−1s−1)

(×10−3 s−1)

(nM)

S4-S

10.7 ± 0.3

0.37 ± 0.06

0.034 ± 0.004

0.13 ± 0.02

0.47 ± 0.02

3.5 ± 0.4

RDE

6.4 ± 0.8

65 ± 3

10 ± 1

Apt1-S

6.7 ± 0.2

7.6 ± 0.6

1.1 ± 0.1

a

N.D.b 0.10 ± 0.01

280 ± 10

2800 ± 200

All experiments were performed in 20 mM sodium phosphate (pH 6.5) with 2 mM magnesium

acetate, 300 mM potassium acetate, or 1 M potassium acetate, 0.1% Tween 20, and 1 mM DTT at 298 K. The kon, koff, and Kd values represent the mean ± SE (n = 3).

b

Not determined for no significant binding.


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Biochemistry

Table 3. Thermodynamics of the binding of S4-S or RDE to RD at 0.3 M potassium acetate a

S4-S

RDE

a

Temperature (K)

N

Kd (nM)

∆G

∆H

(kcal mol−1)

288

0.96 ± 0.03

3± 2

293

0.89 ± 0.03

298

(kcal mol−1)

(kcal mol−1 K−1)

−11.2 ± 0.3

−45.4 ± 0.2

−34.2 ± 0.2

−0.82 ± 0.14

5±3

−11.2 ± 0.4

−51.4 ± 0.4

−40.2 ± 0.8

0.87 ± 0.02

5±3

−11.4 ± 0.4

−56 ± 1

−45 ± 1

303

0.90 ± 0.03

6±3

−11.4 ± 0.3

−57.5 ± 0.8

−46 ± 1

288

1.02 ± 0.04

20 ± 4

−10.2 ± 0.1

−21.1± 0.4

−11.0 ± 0.3

293

0.93 ± 0.02

28 ± 2

−10.13 ± 0.03

−23.69 ± 0.02

−13.56± 0.04

298

0.99 ± 0.08

66 ± 5

−9.82 ± 0.05

−25.5 ± 0.4

−15.7 ± 0.4

303

0.87 ± 0.08

130 ± 30

−9.6 ± 0.2

−29 ± 1

−20 ± 1

−0.52 ± 0.05

All experiments were performed in 20 mM sodium phosphate (pH 6.5) with 2 mM magnesium acetate, 300 mM potassium

acetate, 10% glycerol, and 1 mM DTT. The N, Kd, ∆G, ∆H, and T∆S values represent the mean ± SE (n = 3). b

∆Cpb

T∆S (kcal mol−1)

The ∆Cp value was obtained between 288 K and 303 K.

ACS Paragon Plus Environment

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Figure Legends Figure 1. The secondary structures of S4, S4-S, RDE, and Apt1-S. The secondary structures of S4 (A) and S4-S (B) were predicted by vs_subopt. Lower-case letters indicate nucleotides derived from the primer-binding sites. The secondary structures of RDE (C) and Apt1-S (D). The important bases for RD binding are highlighted in bold.

Figure 2. Effect of salt concentration on S4-S binding to RD. SPR sensorgrams of S4-S/RD interaction at RD concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 nM with 0.3 M potassium acetate (A) and at RD concentrations of 37.5, 75, 150, 300, 600, and 1200 nM with 1 M potassium acetate (D). SPR sensorgrams of RDE/RD interaction at RD concentrations of 2.5, 5, 10, 20, 40, and 80 nM with 0.3 M potassium acetate (B) and at an RD concentration of 1000 nM with 1 M potassium acetate (E). SPR sensorgrams of Apt1-S/RD interaction at RD concentrations of 2.5, 5, 10, 20, 30, and 40 nM with 0.3 M potassium acetate (C) and at RD concentrations of 150, 300, 600, 900, 1200, and 2400 nM with 1 M potassium acetate (F).

Figure 3. ITC data for S4-S/RD interaction and RDE/RD interaction. Titrations of RD with S4-S (A) and with RDE (B) were carried out at 298 K. Top panels show ITC traces and bottom panels show integrated heat values. Data were fitted using the one set of sites binding model.

Figure 4. Temperature dependence of enthalpy for S4-S binding and RDE binding to RD. ∆H values a given temperature for S4-S (open) and RDE (filled). Error bars show the standard error of the mean. These plots provide ∆Cp values for S4-S/RD and RDE/RD interactions.


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Biochemistry

Figure 5. Graphical view of enthalpy−entropy partition. Comparison of thermodynamic parameters for S4-S/RD interaction (gray bars) and RDE/RD interaction (black bars) at 0.3 M potassium acetate at 298 K indicates different partitioning of free energy into enthalpy and entropy terms.


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Biochemistry

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For Table of Contents Use Only Title “Kinetic and thermodynamic analyses of interaction between a high-affinity RNA aptamer and its target protein” Authors Ryo Amano, Kenta Takada, Yoichiro Tanaka, Yoshikazu Nakamura, Gota Kawai, Tomoko Kozu, and Taiichi Sakamoto


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. The secondary structures of S4, S4-S, RDE, and Apt1-S. The secondary structures of S4 (A) and S4-S (B) were predicted by vs_subopt. Lower-case letters indicate nucleotides derived from the primerbinding sites. The secondary structures of RDE (C) and Apt1-S (D). The important bases for RD binding are highlighted in bold. 294x414mm (300 x 300 DPI)


Biochemistry

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Figure 2. Effect of salt concentration on S4-S binding to RD. SPR sensorgrams of S4-S/RD interaction at RD concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 nM with 0.3 M potassium acetate (A) and at RD concentrations of 37.5, 75, 150, 300, 600, and 1200 nM with 1 M potassium acetate (D). SPR sensorgrams of RDE/RD interaction at RD concentrations of 2.5, 5, 10, 20, 40, and 80 nM with 0.3 M potassium acetate (B) and at an RD concentration of 1000 nM with 1 M potassium acetate (E). SPR sensorgrams of Apt1-S/RD interaction at RD concentrations of 2.5, 5, 10, 20, 30, and 40 nM with 0.3 M potassium acetate (C) and at RD concentrations of 150, 300, 600, 900, 1200, and 2400 nM with 1 M potassium acetate (F). 412x373mm (300 x 300 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. ITC data for S4-S/RD interaction and RDE/RD interaction. Titrations of RD with S4-S (A) and with RDE (B) were carried out at 298 K. Top panels show ITC traces and bottom panels show integrated heat values. Data were fitted using the one set of sites binding model. 367x279mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Temperature dependence of enthalpy for S4-S binding and RDE binding to RD. ∆H values a given temperature for S4-S (open) and RDE (filled). Error bars show the standard error of the mean. These plots provide ∆Cp values for S4-S/RD and RDE/RD interactions. 294x216mm (300 x 300 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Graphical view of enthalpy−entropy partition. Comparison of thermodynamic parameters for S4S/RD interaction (gray bars) and RDE/RD interaction (black bars) at 0.3 M potassium acetate at 298 K indicates different partitioning of free energy into enthalpy and entropy terms. 311x213mm (300 x 300 DPI)


Kinetic and Thermodynamic Analyses of ... - ACS Publications

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