Fast complementation of split fluorescent protein triggered by DNA hybridization.
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.
Demidov
Vadim V
VV
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
Dokholyan
Nikolay V
NV
Witte-Hoffmann
Carlos
C
Chalasani
Poornima
P
Yiu
Hung-Wei
HW
Ding
Feng
F
Yu
Yong
Y
Cantor
Charles R
CR
Broude
Natalia E
NE
D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D017510:Protein Folding; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence / Q000379:methods*
D009841:Oligonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA