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. 2009 Jul 15;19(14):3748-51.
doi: 10.1016/j.bmcl.2009.04.141. Epub 2009 May 5.

Systematic Evaluation of Split-Fluorescent Proteins for the Direct Detection of Native and Methylated DNA

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Free PMC article

Systematic Evaluation of Split-Fluorescent Proteins for the Direct Detection of Native and Methylated DNA

Jennifer L Furman et al. Bioorg Med Chem Lett. .
Free PMC article

Abstract

In order to directly detect nucleic acid polymers, we have designed biosensors comprising sequence-specific DNA binding proteins tethered to split-reporter proteins, which generate signal upon binding a predetermined nucleic acid target, in an approach termed SEquence-Enabled Reassembly (SEER). Herein we demonstrate that spectroscopically distinct split-fluorescent protein variants, GFPuv, EGFP, Venus, and mCherry, function effectively in the SEER system, providing sensitive DNA detection and the ability to simultaneously detect two target oligonucleotides. Additionally, a methylation-specific SEER-Venus system was generated, which was found to clearly distinguish between methylated versus non-methylated target DNA. These results will aid in refinement of the SEER system for the detection of user defined nucleic acid sequences and their chemical modifications as they relate to human disease.

Figures

Figure 1
Figure 1
SEquence-Enabled Reassembly. The FP fusion constructs are initially non-fluorescent. The introduction of target DNA results in ZF binding, which induces reassembly of a productively fluorescent FP.
Figure 2
Figure 2
Properties of FPs utilized in SEER. The left panels display chromophore structures and wavelengths of maximum excitation (λex) and emission (λem) for each of the full length FP variants. Mutations (A. victoria numbering) are listed below the corresponding structures with chromophore mutations indicated in bold. mCherry includes additional GFP-type residues on both its N- and C-terminus. The right panels show a DNA titration for each SEER-FP system. The bottom panels show specificity data and a DNA titration for the mCpG-SEER system.
Figure 3
Figure 3
Mixed complementation of FPs. (A) A mixture of NVenus-Zif268, CGFPuv-PBSII, and CVenus-PE8B bind their respective targets. Wavelengths of maximum excitation (λex) and emission (λem) are indicated for each reassembled species. (B) NVenus-Zif268, CGFPuv-PBSII, and CVenus-PE8B were mixed in the presence of target DNA, and fluorescence was monitored at the indicated wavelengths. (C) SEER-Venus/Cerulean mixed complementation. The three constructs, NVenus-Zif268, CCerulean-PBSII, and CVenus-PE8B, bind their cognate sequences. Wavelengths of maximum excitation and emission are indicated next to the reassembled FPs. (D) NVenus-Zif268, CCerulean-PBSII, and CVenus-PE8B were mixed in the presence of target DNA, and fluorescence was monitored at the indicated wavelengths.

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