Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2021 Apr 3

PMID 33807659

Citations 1

Authors

Nakshi Desai

Viraj Shah

Bhaskar Datta

Affiliations

Soon will be listed here.

Abstract

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.

Citing Articles

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions.

Santos T, Salgado G, Cabrita E, Cruz C Pharmaceuticals (Basel). 2021; 14(8).

PMID: 34451866 PMC: 8401999. DOI: 10.3390/ph14080769.

References

Wang S, Su H, Gu Y, Lin S, Tan J, Huang Z . Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance. Biochem Biophys Rep. 2017; 5:439-447. PMC: 5600415. DOI: 10.1016/j.bbrep.2015.09.022. View

Laguerre A, Hukezalie K, Winckler P, Katranji F, Chanteloup G, Pirrotta M . Visualization of RNA-Quadruplexes in Live Cells. J Am Chem Soc. 2015; 137(26):8521-5. DOI: 10.1021/jacs.5b03413. View

Thapar R, Wang J, Hammel M, Ye R, Liang K, Sun C . Mechanism of efficient double-strand break repair by a long non-coding RNA. Nucleic Acids Res. 2020; 48(19):10953-10972. PMC: 7641761. DOI: 10.1093/nar/gkaa784. View

Ma D, Zhang Z, Wang M, Lu L, Zhong H, Leung C . Recent Developments in G-Quadruplex Probes. Chem Biol. 2015; 22(7):812-28. DOI: 10.1016/j.chembiol.2015.06.016. View

De Magis A, Manzo S, Russo M, Marinello J, Morigi R, Sordet O . DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proc Natl Acad Sci U S A. 2018; 116(3):816-825. PMC: 6338839. DOI: 10.1073/pnas.1810409116. View

Han H, Hurley L, Salazar M . A DNA polymerase stop assay for G-quadruplex-interactive compounds. Nucleic Acids Res. 1998; 27(2):537-42. PMC: 148212. DOI: 10.1093/nar/27.2.537. View

Zhang S, Sun H, Yang D, Liu Y, Zhang X, Chen H . Evaluation of the selectivity of G-quadruplex ligands in living cells with a small molecule fluorescent probe. Anal Chim Acta X. 2020; 2:100017. PMC: 7587023. DOI: 10.1016/j.acax.2019.100017. View

Jaubert C, Bedrat A, Bartolucci L, Di Primo C, Ventura M, Mergny J . RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand. Sci Rep. 2018; 8(1):8120. PMC: 5970142. DOI: 10.1038/s41598-018-26582-3. View

Nagesh N, Buscaglia R, Dettler J, Lewis E . Studies on the site and mode of TMPyP4 interactions with Bcl-2 promoter sequence G-Quadruplexes. Biophys J. 2010; 98(11):2628-33. PMC: 2877325. DOI: 10.1016/j.bpj.2010.02.050. View

10.

Brooks T, Kendrick S, Hurley L . Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS J. 2010; 277(17):3459-69. PMC: 2971675. DOI: 10.1111/j.1742-4658.2010.07759.x. View

11.

Ragazzon P, Chaires J . Use of competition dialysis in the discovery of G-quadruplex selective ligands. Methods. 2007; 43(4):313-23. PMC: 2171366. DOI: 10.1016/j.ymeth.2007.08.003. View

12.

Rhodes D, Lipps H . G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 2015; 43(18):8627-37. PMC: 4605312. DOI: 10.1093/nar/gkv862. View

13.

Arnoult N, Shin-Ya K, Londono-Vallejo J . Studying telomere replication by Q-CO-FISH: the effect of telomestatin, a potent G-quadruplex ligand. Cytogenet Genome Res. 2009; 122(3-4):229-36. DOI: 10.1159/000167808. View

14.

Xu S, Li Q, Xiang J, Yang Q, Sun H, Guan A . Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes. Sci Rep. 2016; 6:24793. PMC: 4838840. DOI: 10.1038/srep24793. View

15.

De Cian A, Guittat L, Shin-Ya K, Riou J, Mergny J . Affinity and selectivity of G4 ligands measured by FRET. Nucleic Acids Symp Ser (Oxf). 2006; (49):235-6. DOI: 10.1093/nass/49.1.235. View

16.

Zhang S, Sun H, Chen H, Li Q, Guan A, Wang L . Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe. Biochim Biophys Acta Gen Subj. 2018; 1862(5):1101-1106. DOI: 10.1016/j.bbagen.2018.01.022. View

17.

Drygin D, Siddiqui-Jain A, OBrien S, Schwaebe M, Lin A, Bliesath J . Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis. Cancer Res. 2009; 69(19):7653-61. DOI: 10.1158/0008-5472.CAN-09-1304. View

18.

Nakanishi C, Seimiya H . G-quadruplex in cancer biology and drug discovery. Biochem Biophys Res Commun. 2020; 531(1):45-50. DOI: 10.1016/j.bbrc.2020.03.178. View

19.

Bhasikuttan A, Mohanty J . Targeting G-quadruplex structures with extrinsic fluorogenic dyes: promising fluorescence sensors. Chem Commun (Camb). 2015; 51(36):7581-97. DOI: 10.1039/c4cc10030a. View

20.

Monchaud D, Allain C, Teulade-Fichou M . Development of a fluorescent intercalator displacement assay (G4-FID) for establishing quadruplex-DNA affinity and selectivity of putative ligands. Bioorg Med Chem Lett. 2006; 16(18):4842-5. DOI: 10.1016/j.bmcl.2006.06.067. View