Degassing station

The isothermal titration calorimetry experiments were conducted using a Nano-ITC calorimeter (TA Instruments). A 500 μM GL1 peptide solution and a 10 μM siRNA solution were both prepared in RNase-free water. All of the samples were degassed in a degassing station (TA Instruments) prior to the experiments. RNase-free water was placed in the ITC reference cell. For each titration, 2 μl of the peptide in a pipette rotating at 250 rpm was injected into the siRNA solution in the sample cell of the calorimeter, which was equilibrated to 25°C, with an interval of 300 s between injections. The heat of dilution was measured by titrating the GL1 solution into RNase-free water and was later subtracted from the sample measurement. The data were analyzed using NanoAnalyze software v.3.1.2.

The nanoITC experiments were performed at 298 K with a nano-isothermal titration calorimeter (TA Instruments, New Castle, USA). All samples were degassed (degassing time t = 15 min) using Degassing Station (TA Instruments, New Castle, USA). Calorimetric measurements were determined based on the experimental and theoretical parameters [15 (link), 16 (link), 25 (link)–28 (link)] (Table 1). All solutions were prepared using a phosphate buffer (0.05 M, pH 7.4).Table 1

Experimental dataset

Property	Value
Syringe concentration (10–3 mol/dm3)	1.2
Cell concentration (10–5 mol/dm3)	3
Initial cell Vol (μl)	300
Inj. interval (s)	180
Inj. volume (μl)	2.38
Temperature (K)	298
Stir rate (RPM)	300

The Gibbs free energy change ΔG has been obtained based on Eq. (2) [29 (link)] $$\mathrm{\Delta }\text{G}=\mathrm{\Delta }\text{H}-\text{T}\mathrm{\Delta }\text{S}\left[\text{kcal}\times {\text{mol}}^{-1}\right],$$ where ΔH is enthalpy change [kcal × mol⁻¹]; T is temperature [K]; ΔS is entropy change [kcal × mol⁻¹ K⁻¹].

Ligands were always freshly prepared in the exact same buffer as was used for the size exclusion run. Samples were degassed, and temperature was equilibrated using a degassing station (TA Instruments). About 400 μl protein sample of different concentrations (Table S5) depending on affinity was transferred into the sample cell of a nanoITC (TA Instruments), and 50 μl of about tenfold concentrated ligand solution was loaded into the injection needle. Multiple injection measurements were carried out at 293 K, 300 rpm stirring rate, and 250 s spacings between each 2 μl injection. The heat quantity past injection was determined by integration of the measured peaks. Every protein–ligand combination was measured as biological triplicate. Subtraction of heat of dilution measurements, peak integration, and one site binding fits were done with NanoAnalyze (TA Instruments). Reported errors are the errors between fits of the measured triplicates.

Pfs230D1M, Pfs230D1A, and 4F12 solutions were prepared for isothermal calorimetry (ITC) experiments by dialysis against PBS for 2 h. Prior to each experiment, the Pfs230D1M, Pfs230D1A, and 4F12 protein solutions were filtered through a Whatman inorganic membrane filter or 0.22 µm filter, checked for the presence of aggregates by dynamic light scattering using Malvern Nano-S instrument, and degassed for 30 min using the TA Instruments degassing station. The solutions containing Pfs230D1M or Pfs230D1A were loaded in the syringe at 165 μM or 49 μM, and 4F12 was placed in the calorimeter cell at 23 μM or 3 μM, respectively. Binding experiments were performed on a TA Instruments Low Volume Nano ITC instrument at 25 °C using 20 injections of 2.5 μL each, with injection interval of 300 s, and a syringe stirring speed of 250 rpm. The initial injections were excluded from the data analyses. The ITC data were fitted to the independent binding site model using the TA Instruments NanoAnalyze software.

Isothermal titration calorimetry (ITC) measurements were performed in a Nano ITC standard cell (TA Instruments, New Castle, USA) at 25°C at a constant stirring rate of 300 rpm. Proteins and aptamer were dialyzed extensively against ITC buffer (20 mM KH₂PO₄/K₂HPO₄ pH 7.4, 50 mM NaCl) prior to any titration experiment. DNA oligonucleotides were hybridized and diluted to working concentrations in ITC buffer. All samples were degassed for 15 min at 900 rpm under vacuum using a degassing station (TA Instruments) prior to the titrations. Titration of either TetR or variant TetR-Q38A to the aptamer was performed with a 175 μM protein (dimeric TetR or TetR-Q38A) and a 15 μM aptamer concentration. Titration of variant TetR-Y42A to the aptamer was performed with 345 μM dimeric TetR-Y42A and 15 μM aptamer. Each experiment consisted of 35 × 5 μl injections with 360 s-long pauses in-between injections. The first injection volume was set to 2 μl to remove mixed reactants in the needle tip resulting from diffusion during the equilibration period of the instrument. A blank titration of the respective TetR variant into ITC buffer was used to account for any heat resulting from mixing and dilution. Data were analyzed using the NanoAnalyze software version 3.7.5 (TA Instruments).

Pfs25M, Pfs25H, and 1G2 IgG solutions were prepared for isothermal titration calorimetry (ITC) experiments by dialysis against PBS for 2 h. Prior to each experiment, the Pfs25M, Pfs25H, and 1G2 protein solutions were filtered through a Whatman inorganic membrane filter or 0.22 µm filter, checked for the presence of aggregates by dynamic light scattering using a Malvern Nano-S instrument, and degassed for 30 min using the TA Instruments degassing station. The solutions containing Pfs25M or Pfs25H were loaded in the syringe at 165 μM or 49 μM, and 1G2 was placed in the calorimeter cell at 23 μM or 3 μM, respectively. Binding experiments were performed on a TA Instruments Low Volume Nano ITC instrument at 25 °C using 20 injections of 2.5 μL each, with injection interval of 300 s, and a syringe stirring speed of 250 rpm. The initial injections were excluded from the data analyses. The ITC data were fitted to the independent binding site model using the TA Instruments NanoAnalyze software.

DSC measurements were performed using a Nano DSC system (TA). Prior to scanning, samples were degassed under vacuum for 15 min using a degassing station (TA). DSC thermograms were determined by monitoring the difference in heat capacity in solution upon increasing temperature at a scan rate of 1 °C min⁻¹ by heating the sample from 15 °C to 75 °C under increased pressure (3 atm). All proteins used in this study were extensively dialyzed against a buffer containing 50 mM NaCl, 50 mM Tris, pH 7.6, and the dialysis buffer was used for instrumental baseline scans and as reference samples. Protein concentrations used were 1.0 mg/ml, corresponding to 75.0 μM for ARID4A/ARID4B TD121 proteins. Data were fitted to a two-state scaled model using NanoAnalyze software.