Fast complementation of split fluorescent protein triggered by DNA hybridization
| Autor: | Carlos Witte-Hoffmann, Natalia E. Broude, Poornima Chalasani, Nikolay V. Dokholyan, Feng Ding, Yong Yu, Hung Wei Yiu, Charles R. Cantor, Vadim V. Demidov |
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| Rok vydání: | 2006 |
| Předmět: |
Protein Folding
Multidisciplinary Oligonucleotide DNA–DNA hybridization Green Fluorescent Proteins Oligonucleotides Nucleic Acid Hybridization DNA Biological Sciences Biology Fluorescence Green fluorescent protein Nucleic acid thermodynamics chemistry.chemical_compound Bimolecular fluorescence complementation Spectrometry Fluorescence chemistry Biochemistry Nucleic acid Biophysics Sequence Deletion |
| Zdroj: | Proceedings of the National Academy of Sciences. 103:2052-2056 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein–protein or protein–nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells. |
| Databáze: | OpenAIRE |
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