Fast complementation of split fluorescent protein triggered by DNA hybridization

*Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, Boston, MA 02215;
^‡Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
^§Sequenom, Inc., San Diego, CA 92121

Contributed by Charles R. Cantor, December 22, 2005

Abstract

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.

Footnotes

^†To whom correspondence may be addressed. E-mail: vvd{at}bu.edu, ccantor{at}sequenom.com, or neb{at}bu.edu
Author contributions: V.V.D., N.V.D., C.W.-H., C.R.C., and N.E.B. designed research; V.V.D., N.V.D., C.W.-H., P.C., H.-W.Y., F.D., and Y.Y. performed research; V.V.D., N.V.D., F.D., C.R.C., and N.E.B. analyzed data; and V.V.D., C.R.C., and N.E.B. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

DMD,

discrete molecular dynamics;

biotin-HPDP,

N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide