Journal: bioRxiv

Article Title: Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

doi: 10.64898/2026.04.01.715772

Figure Lengend Snippet: a, CLSM images of glycoRNAs on the surface of MCF-7 cells with COMPASS and various control groups lacking components of the COMPASS. w/o, without. Scale bar, 100 μm. b, Flow cytometric analysis of COMPASS-processed MCF-7 cells incubated with Ac 4 ManNAz, 5-EU, or both, followed by sequential treatment with DBCO-AF647 and N 3 -AF488. c, RT-qPCR analysis of RNA samples extracted from DTWD2 gene silencing by siRNA and the negative control (NC). d, CLSM images of MCF-7 cells after silencing of the DTWD2 gene by siRNA or NC. Scale bar, 20 μm. e, Quantification of average fluorescence intensity in d . Data in e is representative of three independent experiments; n = 5 frames. f, Colocalization images (overlay channel) of glycoRNAs (COMPASS channel) with plasma membrane (DiD channel) or lipid rafts (CT-B channel). Scale bar, 10 μm. The line diagram depicts the intensity profiles of the fluorescent signals along the yellow arrows in the overlay images. The Pearson scatterplot on the right illustrates the degree of colocalization in the overlay image. Data are shown as mean ± s.d. Statistical significance was analyzed by unpaired t-test; NS, not significant;(*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001.

Article Snippet: Subsequently, DMB labeling was performed with the Sialic Acid Fluorescence Labeling Kit (Takara) and analyzed by HPLC with fluorescence detection.

Techniques: Control, Incubation, Quantitative RT-PCR, Negative Control, Fluorescence, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

doi: 10.64898/2026.04.01.715772

Figure Lengend Snippet: a, Specificity for COMPASS to image glycoRNAs on the MCF-7 cell surface. Imaging with COMPASS after treating MCF-7 cells with two RNases (RNase A and RNase T1), two glycosidases (PNGase F and NA), two glycosylation inhibitors (tunicamycin and BG), or two proteases (trypsin and proteinase K), respectively. Scale bar, 20 μm. b, Quantification of average fluorescence intensity in a . Data in b is representative of three independent experiments; n = 5 frames. Data are shown as mean ± s.d. Statistical significance was analyzed by unpaired two-tailed Student’s t-test; NS, not significant;(*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001. c, Generality for COMPASS to image glycoRNAs on the cell surface of Hela cells, HepG2 cells, LM3 cells, and IMR-32 cells. Scale bar, 20 μm.

Article Snippet: Subsequently, DMB labeling was performed with the Sialic Acid Fluorescence Labeling Kit (Takara) and analyzed by HPLC with fluorescence detection.

Techniques: Imaging, Glycoproteomics, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

doi: 10.64898/2026.04.01.715772

Figure Lengend Snippet: a, CLSM images of glycoRNAs by COMPASS in MCF-7 and MCF-10A cells. Scale bar, 100 μm. b, Quantification of the average fluorescence intensity of the FRET channel in a . Data in b is representative of three independent experiments; n = 5 frames. Data are shown as mean ± s.d. c, Schematic illustration of the malignancy transformation model, in which MCF-10A cells were exposed to DMBA to induce malignant transformation. d, Schematic illustration of chemotherapy model, in which MCF-7 cells were treated with PTX to induce cell death. e, Visualization of glycoRNAs abundance after treating MCF-10A cells with DMBA. Scale bar, 20 μm. f, Visualization of glycoRNAs after treating MCF-7 cells with PTX. Scale bar, 20 μm. g, Quantification of the average fluorescence intensity of the FRET channel in e . Data in g is representative of three independent experiments; n = 5 frames. Data are shown as mean ± s.d. h, The expression levels of total glycoRNA in MCF-10A cells treated with different concentrations of DMBA were assessed by MCR-free RNA blotting. i, Quantification of the average fluorescence intensity of the FRET channel in f . Data in i is representative of three independent experiments; n = 5 frames. Data are shown as mean ± s.d. j, The expression levels of total glycoRNA in MCF-7 cells treated with different concentrations of PTX were assessed by MCR-free RNA blotting. Statistical significance was analyzed by unpaired t-test; NS, not significant;(*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001.

Article Snippet: Subsequently, DMB labeling was performed with the Sialic Acid Fluorescence Labeling Kit (Takara) and analyzed by HPLC with fluorescence detection.

Techniques: Fluorescence, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

doi: 10.64898/2026.04.01.715772

Figure Lengend Snippet: a, Schematic illustration of the experimental procedure for imaging mouse tissues using COMPASS. Balb/c mice (n=3) were i.v. injected with 4T1 cells in the model groups, while PBS was injected in the control group. The 5-EU (20 mg/ml) was i.p. injected initially, followed by Ac 4 ManNAz (5 mg/kg) once daily for the following three days. b, Images of the lung harvested from mice. Metastatic nodules in the model group are indicated by black circles. Scale bar, 2 mm. c, Hematoxylin & eosin (H&E) staining of tissues from the major organs of control and model mice. Metastatic nodules in the model group are indicated by black circles. Scale bar, 100 µm. d, CLSM images of FRET signals in lung tissues from different treated mice captured with the assistance of COMPASS. The experimental mice were divided into the following two groups (n = 3 per group): a control group and the model group. To establish tumor metastasis models, 4T1 cells (1×10 7 cells) suspended in PBS were injected intravenously through the tail vein in the model group. Meanwhile, the control group was injected with PBS. Mice were labeled with a single or two MCRs (100 μl of 20 mg/ml 5-EU, 0.16 mmole/kg DBCO-AF647), respectively, and subsequent slices were subjected to two-step click chemistry to achieve AF488 and AF647 labeling. Scale bar, 100 µm. e, Intensity surface plots of FRET signals in lung tissues from different treated mice captured with the assistance of COMPASS. f, Quantification of the average fluorescence intensity of nodule versus perinodule regions in the FRET channel on the right side of e . Data in f is representative of three independent experiments; n = 5 frames. Data are shown as mean ± s.d. Statistical significance was analyzed by unpaired t-test; NS, not significant;(*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001.

Article Snippet: Subsequently, DMB labeling was performed with the Sialic Acid Fluorescence Labeling Kit (Takara) and analyzed by HPLC with fluorescence detection.

Techniques: Imaging, Injection, Control, Staining, Labeling, Fluorescence