Antisense Oligonucleotides Used to Identify Telomeric G-Quadruplexes in Metaphase Chromosomes and Fixed Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Foci.

Ting-Yuan Tseng,Chiung-Lin Wang,Ta-Chau Chang,Shin-Ya Liu

doi:10.3390/molecules25184083

Abstract

Identification of the existence of G-quadruplex (G4) structure, from a specific G-rich sequence in cells, is critical to the studies of structural biology and drug development. Accumulating evidence supports the existence of G4 structure in vivo. Particularly, time-gated fluorescence lifetime imaging microscopy (FLIM) of a G4 fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), was used to quantitatively measure the number of G4 foci, not only in different cell lines, but also in tissue biopsy. Here, circular dichroism spectra and polyacrylamide gel electrophoresis assays show that the use of antisense oligonucleotides unfolds their G4 structures in different percentages. Using antisense oligonucleotides, quantitative measurement of the number of o-BMVC foci in time-gated FLIM images provides a method for identifying which G4 motifs form G4 structures in fixed cells. Here, the decrease of the o-BMVC foci number, upon the pretreatment of antisense sequences, (CCCTAA)3CCCTA, in fixed cells and at the end of metaphase chromosomes, allows us to identify the formation of telomeric G4 structures from TTAGGG repeats in fixed cells.

Highlights

Telomeres, the ends of chromosomes, are essential for the integrity of eukaryotic chromosomes [1,2].Human telomeres generally contain many tandem repeats of the TTAGGG/CCCTAA duplex together with a short 30 -overhang of G-rich single-stranded sequence, with 100–200 bases of (TTAGGG)repeats [3,4,5]
polyacrylamide gel electrophoresis (PAGE) assays and Circular dichroism (CD) spectra were used to investigate whether the G4 structures of HT23 and
The PAGE assays clearly showed the unfolding of a large percentage of HT23 G4 structures and the unfolding of a small percentage of MYC22 G4 structures after the overnight addition of their corresponding antisense sequences (Figure 1B)

Summary

Introduction

The ends of chromosomes, are essential for the integrity of eukaryotic chromosomes [1,2].Human telomeres generally contain many tandem repeats of the TTAGGG/CCCTAA duplex together with a short 30 -overhang of G-rich single-stranded sequence, with 100–200 bases of (TTAGGG)repeats [3,4,5]. The ends of chromosomes, are essential for the integrity of eukaryotic chromosomes [1,2]. Human telomeres generally contain many tandem repeats of the TTAGGG/CCCTAA duplex together with a short 30 -overhang of G-rich single-stranded sequence, with 100–200 bases of (TTAGGG). It is found that such G-rich sequences can rapidly form G-quadruplex (G4) structures via Hoogsteen hydrogen bonding in the presence of Na+ or K+ cations [6,7,8]. The G4 formation at the 30 end of the chromosome could inhibit telomerase activity and prevent telomeric elongation [9], suggesting that telomeric G4 structure is a therapeutic target [10,11,12]. Schaffitzel et al [15] used in vitro generated antibodies to show that the ciliate

Results

Discussion

Conclusion