Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2

Significance Discovery of ligands for G protein–coupled receptors (GPCRs) is of importance in receptor biology and pharmacology but is still a challenging issue. Here, we propose a method for the discovery of ligands against GPCRs by employing a virus-like particle (VLP) and show unique properties of identified nucleic acid aptamers for GPCR. One aptamer raised against purinergic receptor P2Y2 (P2RY2), a GPCR, behaves like a partial agonist to unliganded receptor, whereas it exhibits a positive allosteric modulator (PAM) activity to liganded receptor. We demonstrate the validity of our aptamer screening method targeting VLP-stabilized GPCR and a unique aptamer with dual function, agonist and PAM, for GPCR, depending on whether the intrinsic ligand is prebound to the receptor.


Supplementary Materials and Methods
Random RNA library as a control As a control RNA sequence, we used chemically synthesized random RNA library (5'-GGG AGA ACT TCG ACC AGA AG [N35] TAT GTG CGC ATA CAT GGA TCC TC-3', where N35 stand for 35 nucleotide random sequence that are fully modified with 2' OMe).
Fluorescent labeling of aptamers and cell counts with flow cytometry for calculation of the aptamers' dissociation constant To estimate dissociation constant (Kd value) of aptamer c37_7-40 and c37_40-74, these focused aptamers and random library were labeled with Alexa647 by using Ulysis Alexa 647 labeling kit (LifeTechnologies) according to the manufacturer's protocols. Before the assessment day, the pHAGE-P2RY2-IZsGreen vector, which encode human wild type P2RY2 tagged with IRES-GFP, was transfected into HEK293 cells with Lipofectamine2000 according to the manufacturer's protocols. Likewise, the pHAGE-EDNRB-IZsGreen vector was transfected into HEK293 cells as negative control cells.
The day after transfection, various amount of each labeled RNA (at 2000, 1000, 500, 250, 125, 62.5, and 0 nM final concentration) were mixed with HEK293 cells over-expressing P2RY2 and EDNRB. The incubation buffer was SELEX buffer supplemented with 1mg/ml yeast tRNA and 1% BSA to reduce non-specific binding of the aptamers. After incubation for 20 min at room temperature, the cells were subjected to centrifugation at 800 xg for 5 min and then supernatant were removed. Collected cells were resuspended with SELEX buffer, and this washing process was repeated by three-times. After washing step, GFP-and Alexa647-double-positive cells were analyzed with BD FACSCant TM II flow cytometer (BD Biosciences, CA, USA). Briefly, the cells were counted until the number of cells (all events) reached 10,000 cells. Using pulse geometry gates (a height, wide, and area of forward scatter (FSC) and side scatter (SSC)), cell clumps were eliminated. After setting the pulse gates, a gate to select GFP-positive cells was further set for examining interaction of the aptamer with GFP-positive cells, which were expected to overexpress P2RY2 and EDNRB, and then, Alexa647(aptamer)-positive cells were counted. After curve fitting with nonlinear regression, Bmax and Kd values of each RNA were analyzed by GraphPad Prism 8.
Free RNA removal efficiency in washing process of VLP-aptamers complexes with ultrafiltration column To achieve immobilization free separation method, we used an ultrafiltration column, Vivaspin 500 centrifugal concentrator MWCO 100kDa (Sartorius), to isolate aptamer complexed with VLP from unbound RNAs. As a model sequence, tRNA from backer's yeast (Roche Diagnosis), whose lengths range from about 70 to 90 bases, was subjected to the evaluation of efficiency of washing process with the column because the length of typical RNA aptamer is close to that of tRNAs. We first prepared 20 g / 20 L and 10 g / 20 L tRNA solutions, which are corresponding to the amount of RNAs used in first and final rounds in our SELEX, respectively. Two micro liters of them were left for electrophoresis, and the remaining tRNAs were transferred into an ultrafiltration column device. After addition of SELEX buffer up to 500 L, the diluted tRNAs was subjected to a centrifugation at 14,000 xg for 5 min. Of the retaining tRNA in the device, two micro liters of the tRNA was saved for electrophoresis, and then a series of dilution process was repeated by four-times (total five times). Saved tRNAs and DynaMarker® RNA Low II (BioDynamics Laboratory Inc. Tokyo, Japan) were mixed with two micro liters of formamide and one micro liter of 6x loading buffer, and then heated at 90 o C for 3 min. After that, those samples and the loading marker were subjected to a denaturing urea polyacrylamide electrophoresis at 6%T. Separating tRNAs were visualized with high-sensitive dye, SYBR™ Gold Nucleic Acid Gel Stain (ThermoFisher). Band intensities of RNAs in each lane was examined by ImageJ software.
NTPs removal efficiency with ultrafiltration column in aptamer purification process To estimate the degree of carryover of NTPs from purified aptamers after IVT (in vitro transcription) to the incubation between aptamers and VLPs, we first prepared NTPs mixture at a final concentration of 2.5 mM in total 100 L without aptamer library, which are same concentration and volume in the purification step of aptamers after IVT. Two micro liters of them were left for absorbance measurement, and the remaining NTP solution was transferred into an ultrafiltration column device. After addition of 400 L of SELEX buffer, the diluted NTPs was subjected to a centrifugation at 14,000 xg for 5 min. Of the retaining NTPs in the device, two micro liters of the NTPs were saved for absorbance measurement, and then a series of dilution process was repeated by fourtimes (total five-times  , approximately 20 and 10 µg of yeast tRNAs were subjected to the same washing procedure as the washing process in SELEX (details were described in Supplementary Materials and Methods). The washing procedure was repeated by five-times, and a portion of each remaining solution in the devices was subjected to denatured PAGE. (B) Visualization of RNAs with fluorescent dye on the gel. The separated tRNAs on the gel were visualized with high-sensitive dye, SYBR Gold gel staining dye. Washings three-times and five-times resulted in the reduction of 85 to 90% and 90 to 95% tRNAs, respectively. . Based on the results, the eight sequences are divided into two major clusters, which are marked in red and blue lines. Because of the highest read number, c1 sequence was selected as candidate that represent a major cluster marked in red line. Subsequently, c11 and c37 were selected as candidates in the other major cluster marked in blue line, because the cluster analysis showed that c11 and c37 is located at the farthest position from c1 sequence. Besides, the sequences c11 and c37 relatively contained many motifs, which was not contained the motif "A" that are involved in all other sequences. Thus, c1, c11, and c37 sequences were selected as candidates and further subjected to the cell-based analysis. 10 0 10 1 10 2 10 3 10 4  S5. Agonist assay of aptamers from first screening. The agonist activity of aptamers c1, c11, and c37, which were selected from first screening, was examined. Chemical agonist PSB1114 was used as a positive control. The aptamers and chemical agonist exposed to the cells overexpressing P2RY2 at final concentrations of 0, 2. 44, 9.77, 39.1, 156.3, 625, 2500, and 4000 nM. The inset is an enlarged view focusing on the aptamers' agonistic activity, which seems not to reach a plateau even at 4000 nM. Data represent the mean ± s.d. of three measurements. The values are expressed as RLU (in %) to the 4000 nM PSB1114 level after subtraction of basal LU in control cells without treatment. EC50 value and 95% CI (confidence internal) of PSB1114 were indicated, but that of the aptamers was unavailable due to their weak activity.   The expression levels of the P2Y family proteins P2RY4 and P2RY11 that are responsive to a common ligand with P2RY2, was confirmed by western blotting. Each expression plasmid encoding P2RY2, P2RY4 and P2RY11 ORF fused with the His6-V5 tag was transfected into HEK293 cells. Cell lysates were prepared 24 h after transfection, and the expression levels of each receptor was examined using anti-V5 antibody. (B) Agonist assay of the aptamers c1, c11 and c37 for P2RY4. Cells transfected with P2RY4 expression plasmid were exposed to the aptamers c1, c11, c37, and UTP (an endogenous agonist for P2RY4). (C) Agonist assay of the aptamers c1, c11 and c37 for P2RY11. Agonist assay as in (B) was carried out by using the aptamers c1, c11, c37, and ATP (an endogenous agonist). Data represent the mean ± s.d. of four independent experiments. The values are expressed as RLU (in %) to the 2.5 mM UTP/ATP level without aptamer after subtraction of basal LU in control cells without treatment. EC50 and 95% CI (confidence interval) of the aptamers and chemicals were shown when those values were available. Inhibitory activity of aptamers c1, c11, and c37 at the indicated concentrations to P2RY4 and P2RY11 was examined. Cells overexpressing P2RY4 and P2RY11 were post-treated by 10 nM UTP and 300 nM ATP, respectively. The values are expressed as RLU to the 10 nM UTP level for P2RY4 and the 300 nM ATP level for P2RY11 without aptamer. Data represent the mean ± s.d. of three independent experiments. The aptamers did not show obvious inhibitory activity to the family receptors. IC50 and 95% CI (confidence interval) values of each aptamer were calculated but they were unavailable due to their weak or no activity. Normalized RLU (%) 10 0 10 1 10 2 10 3 10 4

Aptamer and chemical [nM]
AR-C118925XX         *4. Five hundred micro liter of SELEX buffer was added to ultrafiltration column and subjected to centrifugation to reduce volume, and then flow through were discarded. This procedure was repeated to remove free RNA sequences from the complexes as a washing process.