Professor Xu Zhangrun’s team from the School of Science of Northeastern University published an online research paper titled “FRET imaging of glycoRNA on small extracellular vesicles enabling sensitive cancer diagnostics” in the international authoritative academic journal Nature Communications. Professor Xu Zhangrun's team proposed a dual-recognition Förster resonance energy transfer technique (drFRET), which for the first time achieved sensitive detection of glycoRNA, a novel cancer marker in biological body fluids, on small extracellular vesicles, providing a new tool for early cancer screening and precise typing. NEU is the institution where the first author of this paper. Ren Tingju, a doctoral student from the Department of Chemistry, School of Science, NEU, is the first author of this paper, and Professor Xu Zhangrun is the sole corresponding author.
In 2021, the team of Professor Carolyn R. Bertozzi from Stanford University (winner of the 2022 Nobel Prize in Chemistry) published a breakthrough achievement in Cell, revealing a new type of biomolecule— glycoRNA—on the cell surface. It was confirmed for the first time that RNA can not only be modified by glycosylation, but also be anchored to the surface of cell membranes to participate in biometric recognition. GlycoRNAs are a type of novel membrane-anchored molecules that combine RNA sequence information and glycan modification. They are composed of small RNAs modified by glycans containing sialic acid and have potential important physiological functions. This type of biomolecules formed by the covalent linkage of RNAs and N-linked sugar chains participates in key physiological processes such as cell adhesion and signal transduction through structures such as N-acetylneuraminic acid (Neu5Ac) at the ends of glycans. Due to the lack of efficient and specific imaging techniques, however, people’s understanding of it remains extremely limited.
To address this issue, Professor Xu Zhangrun's research team has developed an in-situ glycoRNA imaging method based on dual-recognition Förster resonance energy transfer. This is the first direct and in situ observation method of glycoRNA on small extracellular vesicles (sEVs), and it has extremely high sensitivity and specificity. The research team first confirmed the presence of Neu5AC-modified glycoRNAs on the surface of sEVs through metabolic labeling and click chemistry techniques, and verified the existence of glycoRNAs with different glycosylation modifications in sEVs. The core of the drFRET technique lies in constructing a "glycan-RNA" dual-recognition probe system to achieve in-situ visualization of glycoRNAs through intermolecular near-field energy transfer. The drFRET technique precisely identifies Neu5Ac at the end of glycoRNAs through the synergistic action of two functionalized DNA probes: a sugar chain recognition probe, designing a DNA aptamer conjugated with Cy3 donor fluorescent dye, and precisely identifying Neu5Ac at the end of glycoRNAs by taking advantage of its spatial folding property; the RNA in situ hybridization probe, designing complementary oligonucleotide chains for the target glycoRNA sequence, labeling the receptor fluorescent dye Cy5, and achieving specific anchoring of RNA components through base pairing. When the two types of probes form a spatial near-field (1-10 nm) on the surface of sEVs, the excited state of Cy3 transfers energy to Cy5 through dipole-dipole coupling, generating a non-radiative energy transfer signal. Spectral crosinterference is eliminated through sensitization emission correction technology to ensure that the signal only originates from the covalent binding sites of glycans and RNAs, effectively avoiding the non-specific interference of traditional single-probe detection.
The research team analyzed sEVs glycoRNAs from 7 cancer cell lines and 100 clinical samples using drFRET technique. Through unsupervised hierarchical clustering analysis of the signals of five glycoRNAs on the surface of sEVs derived from serum, completely separated clusters were formed between cancer and non-cancer samples, and both the diagnostic sensitivity and specificity reached 100% (95% CI). A precise cancer typing model was further constructed through principal coordinate analysis, and the overall typing accuracy rate of the six cancer types reached 89% (95% CI). Studies have shown that the abnormal expression of glycoRNAs on the surface of tumor sEVs is highly correlated with cancer progression, which provides a brand-new molecular target for liquid biopsy of cancer. The research also revealed that glycoRNAs might mediate the adhesion and endocytosis process of sEVs to target cells through sugar-protein interactions, and ultimately play a key role in the delivery of information by tumor cells to distant organs and the construction of the pre-metastasis microenvironment.
This research was supported by the National Natural Science Foundation of China. Dr. Zhang Yingzhi from the First Affiliated Hospital of China Medical University/National Clinical Research Center for Medical Laboratory Science, Associate Professors Wang Yue and Yang Chunguang from the Department of Chemistry, College of Science, NEU, were also involved in this work. Professor Wang Jianhua, Professor Yu Yongliang and others have provided strong support and assistance for this work.
