Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Zhaotong Wang,Tetsuhiro Nemoto,Takamasa Mizoguchi,Hiroyuki Nakamura,Masaya Nakajima,Yuka Sakamoto,Motoyuki Itoh,Takahito Kuribara,Mayuu Iwata,Toshihiko Murayama

doi:10.1098/rsob.200241

Abstract

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Highlights

Distamycin is a small antibiotic molecule containing tri-N-methylpyrrole (Py) that binds to the AT-rich DNA minor groove [1]
We depict the synthesis of Py3-FITC and its 1H-Nuclear magnetic resonance (NMR) map in detail
We examined Py3-FITC staining from the 1-cell stage to the shield stage (6 hpf )

Summary

Introduction

Distamycin is a small antibiotic molecule containing tri-N-methylpyrrole (Py) that binds to the AT-rich DNA minor groove [1]. Poly-Py-based polyamides are used as sequence-specific DNA probes [2]. Fluorescence-conjugated poly-Py-based polyamides are thought to be useful as nuclear-specific probes. Several factors influence the nuclear uptake of poly-Pybased polyamides [3]. Best et al [3] demonstrated that poly-Py-based polyamides with good nuclear uptake affinity have several principal features, such as an eight-ring polyamide DNA-binding domain, one or more positive charges, and a conjugated fluorescein fluorophore. Long-length poly-Pybased polyamides are not always effective. A moderate length of fluorophore polyamide has many advantages: increasing stabilization, increasing binding intensity, cell nuclear uptake and protein affinity. The localization of fluorescence-conjugated polypyrroles in living animals is unclear. We used three types of FITC-conjugated compounds to assess the effect of pyrrole number on their localization in living animals

Methods

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Discussion

Conclusion