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阳离子化明胶与分子信标复合物的设计用于可视化细胞内 mRNA。

Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA.

机构信息

Laboratory of Biomaterials, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.

出版信息

PLoS One. 2021 Jan 25;16(1):e0245899. doi: 10.1371/journal.pone.0245899. eCollection 2021.

DOI:10.1371/journal.pone.0245899
PMID:33493232
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7833158/
Abstract

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

摘要

本研究旨在制备阳离子化明胶-分子信标（MB）复合物，以可视化细胞内信使 RNA（mRNA）。复合物是由不同程度阳离子化的阳离子化明胶和不同比例的 MB 与阳离子化明胶混合制备而成。复合物的表观粒径几乎相似，而zeta 电位则在复合物之间有所不同。无论制备条件如何，复合物在杂交中对靶寡核苷酸具有序列特异性。细胞毒性和复合物进入细胞的量随阳离子化程度和阳离子化明胶浓度的增加而增加。与最高程度阳离子化的阳离子化明胶制备的复合物孵育后，检测到高荧光强度。另一方面，以 20 pmole/μg 的比例制备的复合物没有显示出任何细胞毒性。该复合物对于可视化内源性存在的甘油醛-3-磷酸脱氢酶（GAPDH）mRNA 最为有效。此外，对于外源性转染的增强型绿色荧光蛋白（EGFP）mRNA，复合物也能够有效地检测到它。因此，结论是可以通过设计的阳离子化明胶-MB 复合物来可视化活细胞中的内源性和外源性 mRNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/9aa3f063e4bd/pone.0245899.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/b9ee1a1eba06/pone.0245899.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/aa1f88a5406a/pone.0245899.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/3c647428b200/pone.0245899.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/e2067748a212/pone.0245899.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/67bfd7aba7ad/pone.0245899.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/7679e506e72c/pone.0245899.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/9aa3f063e4bd/pone.0245899.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/b9ee1a1eba06/pone.0245899.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/aa1f88a5406a/pone.0245899.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/3c647428b200/pone.0245899.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/e2067748a212/pone.0245899.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/67bfd7aba7ad/pone.0245899.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/7679e506e72c/pone.0245899.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/792f/7833158/9aa3f063e4bd/pone.0245899.g007.jpg

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