Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA.
While there are many hybridization-based DNA sensors, few can detect native double-stranded DNA (dsDNA), which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers with an N-terminal dimerization motif and a C-terminal GFP variant to detect specific dsDNA sequence in a homogeneous solution. When a pair of purified zinc finger-GFP colour variant proteins (Zif12-eCFP, Zif12-eYFP) were mixed and added with specific dsDNA with 12 bp inverted repeat sequence, fluorescence spectra of the solution showed significant concentration-dependent enhancement of fluorescence resonance energy transfer (FRET), with the detection limit of approximately 10 nM. No significant change in FRET was observed upon addition of dsDNA with non-specific sequence, indicating dsDNA-dependent dimerization of the two proteins. This dimerization-based dsDNA sensors will have a range of applications where conventional hybridization-based assay is difficult.
Yoshitake
Kazutoshi
K
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Waki
Shoko
S
Ueda
Hiroshi
H
D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008667:Metalloproteins; D004247:DNA