DOI: 10.5061/dryad.8931zcs1s
title: G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication

description: Future pandemic threats may be caused by novel coronaviruses and influenza
 A viruses. Here we show that when directly added to a cell culture,
 guanosine 12mer (G12) and its phosphorothioate-linked derivatives
 (G12(S)), rapidly entered cytoplasm and suppressed the propagation of
 human coronaviruses and influenza A viruses to between 1/100 and nearly
 1/1000 of normal virus infectivity without cellular toxicity and induction
 of innate immunity. Moreover, G12(S) alleviated the weight loss caused by
 coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with
 left-handed parallel-stranded G-quadruplex, and inhibit the replication
 process by impeding interaction between viral nucleoproteins and viral RNA
 in the cytoplasm. Unlike previous antiviral strategies that target the
 G-quadruplexes of the viral genome, we now show that excess exogenous
 G-quadruplex-forming small RNA displaces genomic RNA from
 ribonucleoprotein, effectively inhibiting viral replication. The approach
 has the potential to facilitate the creation of versatile middle-molecule
 antivirals featuring lipid nanoparticle-free delivery.
description: <strong>QUMA-1 stain for the cells</strong>
 A549 cells were cultured in 96-well plates (ViewPlate). After
 being washed with MEM, cells were cultured in the presence of 2 μM of each
 PS-RNA for 17 h. Cells were then washed with MEM and fixed with 4%
 paraformaldehyde in PBS for 15 min on ice. After washing three times with
 PBS, the cells were observed after 7 h or later by adding PBS containing
 50 nM QUMA-1 and 1 μg/mL DAPI. Confocal imaging was performed using LSM900
 (Zeiss) equipped with Airyscan 2 using Plan-Apochromat 63x/1.40 Oil DIC
 M27 (objective). Excitation and detection wavelengths were 353 nm laser
 and 400-564 nm for DAPI, 612 nm laser and 590-700 nm (AF610) for QUMA-1.
 Images of 8 slices (7 µm thick) were processed by Airyscan-2 were
 superimposed by ZEN (Zeiss). TIFF files (16 bit) were exported and
 intensity values of QUMA-1 fluorescence were quantitated using Fiji
 (ImageJ 2.14.0). To quantify PS-RNA specific QUMA-1 fluorescence, Image-J
 (Fiji) was used to set the threshold at approximately 4000 to exclude
 autofluorescence (lower panel). The DAPI stained area of each image was
 quantified and divided by the number of cells to calculate the area per
 cell. The brightness value per cell was divided by this value to correct
 for the cell size between the images.
description: # G-quadruplex-forming small RNA inhibits coronavirus and influenza A
 virus replication
 [https://doi.org/10.5061/dryad.8931zcs1s](https://doi.org/10.5061/dryad.8931zcs1s) ## Principle Investigator Contact Information Name: Shusuke Kuge Institution: Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University. e-mail: [skuge@tohoku-mpu.ac.jp](mailto:skuge@tohoku-mpu.ac.jp) **QUMA-1 stain for the cells** A549 cells were cultured in 96-well plates (ViewPlate). After being washed with MEM, cells were cultured in the presence of 2 μM of each PS-RNA for 17 h. Cells were then washed with MEM and fixed with 4% paraformaldehyde in PBS for 15 min on ice. After washing three times with PBS, the cells were observed after 7 h or later by adding PBS containing 50 nM QUMA-1 and 1 μg/mL DAPI. Confocal imaging was performed using LSM900 (Zeiss) equipped with Airyscan 2 using Plan-Apochromat 63x/1.40 Oil DIC M27 (objective). Excitation and detection wavelengths were 353 nm laser and 400-564 nm for DAPI, 612 nm laser and 590-700 nm (AF610) for QUMA-1. Images of 8 slices (7 µm thick) were processed by Airyscan-2 were superimposed by ZEN (Zeiss). TIFF files (16 bit) were exported and intensity values of QUMA-1 fluorescence were quantitated using Fiji (ImageJ 2.14.0). To quantify PS-RNA specific QUMA-1 fluorescence, Image-J (Fiji) was used to set the threshold at approximately 4000 to exclude autofluorescence (lower panel). The DAPI stained area of each image was quantified and divided by the number of cells to calculate the area per cell. The brightness value per cell was divided by this value to correct for the cell size between the images.   ### Files and variables We have submitted our raw data for Figure_8g (**Figure_8g_data.csv**) and each of the original TIF files of DAPI (DNA) stain and QUMA-1 (G4 RNA) stain (**Data_FIgure_8g/**), and raw data for Figure_9e (**Figure_9e_data.csv**) and each of the original TIF files for DAPI (DNA) stain and QUMA-1 (G4 RNA) stain (**Data_FIgure_9e/**). ## Description of the data and file structure ### **Figure\_8g\_data.csv**\_data * *Sample_Name*: Name of Sample TIF file * *Size_1*: Number of cells with full DAPI (nucleus) image * *Size_&gt;1/2*: Number of cells with more than half size but not full size of DAPI (nucleus) image * *Size_&lt;1/2*: Number of cells with less than half size of DAPI (nucleus) image * *Cell_No*: Number of full size cells caluculated from number of cells less than and more than half size * *DAPI_area*: DAPI_area of each TIF images * *DAPI_are/Cell_No*: DAPI_area divided by the cell number estimated with size_1, size&gt;1/2 and size&lt;1/2 * *IntDen*: Integrated density of each of QUMA-1 TIF iamge * *IntDen/cell/DAPI_area*: IntDen was divided by cell_number and DAPI-area; In order to normalize cell volume, QUMA-1 fluorescence levels per cell were averaged by cell size. * *Ratio*: each IntDen/cell/DAPI_area was devided by an average of four controls (lines 3 to 5: 16.1536) ### **Figure\_9e\_data.csv**\_data * *Sample_Name*: Name of Sample TIF file * *Size_1*: Number of cells with full DAPI (nucleus) image * *Size_&gt;1/2*: Number of cells with more than half size but not full size of DAPI (nucleus) image * *Size_&lt;1/2*: Number of cells with less than half size of DAPI (nucleus) image * *Cell_No*: Number of full size cells caluculated from number of cells less than and more than half size * *DAPI_area*: DAPI_area of each TIF images * *DAPI_are/Cell_No*: DAPI_area divided by the cell number estimated with size_1, size&gt;1/2 and size&lt;1/2 * *IntDen*: Integrated density of each of QUMA-1 TIF iamge * *IntDen/cell/DAPI_area*: IntDen was divided by cell_number and DAPI-area; In order to normalize cell volume, QUMA-1 fluorescence levels per cell were averaged by cell size. * *Ratio*: each IntDen/cell/DAPI_area was devided by an average of four controls (lines 3 to 6: 0.25) ### **Dataset\_Figure8g/**\_DataFolder **Description:** A549 cells were treated with 2 μM PS-RNAs (indicated in the figures) and stained with QUMA-1. We measured G-quadruplex levels in cells (7 μm thick) by overlaying eight slices acquired with a super-resolution confocal laser scanning microscope (using an LSM900 with Airyscan 2) using the G-quadruplex-specific fluorescent dye QUMA-1. Shown are separate images (f) overlay images (e) of eight planes (z-direction) of QUMA-1 (yellow) and DAPI staining (blue). AF610 (Excitation = 612 nm, Emission = 630 nm, Detection = 590–700 nm) used for QUMA-1 observation. Black and white of AF610 set at approximately 4000 and 12000 (f). **Sample_Name/**: Name of Sample TIF DataFolder containing the following: * **Sample_Name_DAPI.TIF**: DAPI image * **Sample_Name_QUMA-1.TIF**: QUMA-1 image * **Sample_Name_DAPI_QUMA-1.TIF**: Marged image of DAPI and QUMA-1 * **Sample_Name_DAPI.xlsx**_Data: DAPI Area data * *Number*: Number of selected area * *Area*: area of selecrted DAPI area (max 2424x2424 piccels) * **Sample_Name_QUMA-1.xlsx**_Data: QUMA-1 IntDen data * *Number*: Number of selected area * *Area*: area of selected QUMA-1 signal * *Mean*: mean brightness * *IntDen*: integrated density of each brightness ### **Dataset\_Figure\_9e/**\_DataFolder **Description:** QUMA-1-stained cells were observed as described above. **Sample_Name/**: Name of Sample TIF DataFolder containing the following: * **Sample_Name_DAPI.TIF**: DAPI image * **Sample_Name_QUMA-1.TIF**: QUMA-1 image * **Sample_Name_DAPI_QUMA-1.TIF**: Marged image of DAPI and QUMA-1 * **Sample_Name_DAPI.xlsx**_Data: DAPI Area data * *Number*: Number of selected area * *Area*: area of selecrted DAPI area (max 2424x2424 piccels) * **Sample_Name_QUMA-1.xlsx**_Data: QUMA-1 IntDen data * *Number*: Number of selected area * *Area*: area of selected QUMA-1 signal * *Mean*: mean brightness ## Access information Other publicly accessible locations of the data: * [https://doi.org/10.1038/s42003-024-07351-7](https://doi.org/10.1038/s42003-024-07351-7)