A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation.

Xiao-Xia Hu,Sheng-Quan Wang,Meng-Hao Jia,Ming-Qing Zhong,Yan Xu,Shi-Quan Gan,Lei Liu,Fei Jiang,Chao-Da Xiao,Xiang-Chun Shen

doi:10.3389/fchem.2021.781198

Xiao-Xia Hu, Sheng-Quan Wang + Show 8 more

Open Access

https://doi.org/10.3389/fchem.2021.781198

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Abstract

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5′-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

Highlights

G-quadruplexes are four-stranded structures found in DNA/RNA sequences that are rich in guanine residues
To test whether OMT can bind to G-quadruplexes, we used the reverse transcriptase (RTase) reaction-based method modified from a previous study to compare the binding of OMT with different G-quadruplexes (Supplementary Figure S1, Supplementary Table S1) (Katsuda et al, 2016)
Molecules binding to the RNA G-quadruplex can efficiently inhibit the elongation of the reverse transcriptase (RTase) reaction, so as to reduce the production of the full-length complementary DNA strand (Hagihara et al, 2010)

Summary

INTRODUCTION

G-quadruplexes are four-stranded structures found in DNA/RNA sequences that are rich in guanine residues. Compared with double-helical DNAs, G-rich RNAs are relatively unconstrained and form G-quadruplexes (Bugaut and Balasubramanian, 2012). Experimental data proved that RNA G-quadruplexes within such regions exerted important regulatory roles for gene expression (Bugaut and Balasubramanian, 2012). We found that OMT could selectively bind to the VEGF RNA G-quadruplex. The results showed that OMT was a highly specific binder for the VEGF RNA G-quadruplex. The results of luciferase assays confirmed the binding of OMT with the VEGF RNA G-quadruplex in cells. SPR and luciferase experiments showed that OMT can recognize the VEGF RNA G-quadruplexes in different conformations. The results may provide a valuable scaffold for further developing the ligand binding–intended G-quadruplex with high specificity, which may serve as the specific inhibitor for VEGF

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION

DATA AVAILABILITY STATEMENT