Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy

Ting-Yuan Tseng,Cheng-Chung Chang,Mei-Ying Lin,Cheng-Hao Chien,Jen-Fei Chu,Ta-Chau Chang,Wei-Chun Huang

doi:10.1117/1.jbo.18.10.101309

Abstract

The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8 ns and that of interaction with the duplex structure of a calf thymus is ∼1.2 ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

Highlights

A large number of guanine-rich (G-rich) sequences are found in the human genome.[1,2,3] The G-rich sequences can form a G-quadruplex (G4) structure in the presence of salts in vitro.[4,5] It is suggested that the G4 structure is important in the human telomere for protecting the ends of chromosomes and in the gene promoter for regulating gene expression
The fluorescence peak of o-bis (1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) shows a slight difference upon interaction with G4 structures at ∼555 nm and with duplexes at ∼545 nm
We have illustrated that an o-BMVC molecule is a better fluorescent probe for more specific binding to G4 DNA than BMVC

Summary

Introduction

A large number of guanine-rich (G-rich) sequences are found in the human genome.[1,2,3] The G-rich sequences can form a G-quadruplex (G4) structure in the presence of salts in vitro.[4,5] It is suggested that the G4 structure is important in the human telomere for protecting the ends of chromosomes and in the gene promoter for regulating gene expression. Some evidences of the presence of G4 structures in vivo have been reported in the ciliate Stylonychia,[6] the promoter of c-myc,[7] the human telomeres,[8] and the promoter of nonselenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx),[9] the actual case in human cells is still in debate.[10]. Hurley et al reported that the sequence d1⁄2ðTG4AG3Þ2TG4AAG2 (PU27) in c-myc gene promoter can form both intramolecular and intermolecular conformations in Kþ solution.[7] NMR and circular dichroism results showed that the monomeric parallel G4 structure is a major conformation for both PU27 and d1⁄2TGðAG3TG4Þ2AA (PU22).[11] In addition, the broad envelope from the imino proton NMR spectra of the human telomeric sequence d1⁄2AG3ðT2AG3Þ3 (HT22) suggested the coexistence of two different G4 structures.[12,13] telomere sequences with slight differences can adopt different

Methods

Results

Conclusion