Method for detection of specific nucleic acids by recombinant protein with fluorescent resonance energy transfer.
Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.
Endoh
Tamaki
T
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Yokohama, 226-8501, Japan.
Funabashi
Hisakage
H
Mie
Masayasu
M
Kobatake
Eiry
E
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005786:Gene Expression Regulation; D015752:Genes, env; D006367:HeLa Cells; D006801:Humans; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*
D009696:Nucleic Acids; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA