Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA.
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.
Furman
Jennifer L
JL
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
Badran
Ahmed H
AH
Shen
Shengyi
S
Stains
Cliff I
CI
Hannallah
Jack
J
Segal
David J
DJ
Ghosh
Indraneel
I
D004247:DNA / Q000032:analysis* / Q000737:chemistry; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D004247:DNA