Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe.

Ta-Chau Chang,Ting-Yuan Tseng,Chiung-Lin Wang,Wei-Chun Huang

doi:10.3390/molecules27010140

Abstract

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.

Highlights

A large number of guanine (G)-rich sequences are found in the human genome [1,2,3]
To elucidate whether this difference is determined by structure or sequence, we further examined the possible G4 formation from exogenous single-stranded Guanine-rich oligonucleotides (GROs) in live cells
fluorescence lifetime imaging microscopy (FLIM) Images of o-BMVCPUfo22r MonitorTiGnAgGt3hTeG4EAxGo3gTeGn4AouAs G4s in3C.0L1-0 Live Cells 2.9 Figure 1A–F show thACeSM14FA11LIM imTaAg(GGeG3sATG)o43TfTGA5(GTG3μAGMG)43To-BMVC 33a..11nd its mixtures32..w28 ith G4 structures belong to 15 μMHTP23U22, AS141TA1G, 3C(TM2AAG3,)3HT23, and 2W.8

Summary

Introduction

A large number of guanine (G)-rich sequences are found in the human genome [1,2,3]. Such sequences may form G-quadruplex (G4) structures by stacking the G-quartets via Hoogsteen hydrogen bonding of four cyclic guanines under physiological conditions [4,5,6]. We first used FLIM to measure the fluorescence lifetimes of the 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC) upon binding to these exogenous G4 structures in live cells to confirm whether the G4 structures can be maintained in their parallel form but not in their non-parallel form in live cells. To elucidate whether this difference is determined by structure or sequence, we further examined the possible G4 formation from exogenous single-stranded GROs in live cells. TGhe3(GTR2OAsGus3e)d3in this work and the cent2er.8peaks of the o-BMVC decay tim-es upon binding

Results

Unfolding of Exogenous CMA G4 Structure by Its Anti-CMA in CL1-0 Live Cells

Chemical Properties of o-BMVC Molecule

Confocal Microscopy

Cell Cultures