Method for Detection of Specific Nucleic Acids by Recombinant Protein with Fluorescent Resonance Energy Transfer
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- 作者：Tamaki Endoh ; Hisakage Funabashi ; Masayasu Mie ; and Eiry Kobatake
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：July 15, 2005
- 年：2005
- 卷：77
- 期：14
- 页码：4308 - 4314
- 全文大小：261K
- 年卷期：v.77,no.14(July 15, 2005)
- ISSN：1520-6882
文摘
Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detectspecific nucleic acids using recombinant protein andoligonucleotides. A recombinant protein YRGnC-11ad,which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescentprotein (ECFP) was constructed and expressed in HeLacells. Rev-peptide, which corresponds to amino acids34-50 of the HIV-1 Rev protein, indicates disorderedstructure in solution but forms 
-helical and elongatedconformation upon binding to Rev response element RNA(RRE-RNA) and Rev-aptamer, respectively. We confirmedthat YRGnC-11ad could specifically bind to RRE-RNA andRev-aptamer in cell lysate, and fluorescent resonanceenergy transfer (FRET) signal was changed upon bindingfollowing the conformational change of Rev-peptide. Toutilize this FRET signal change toward the detection ofspecific nucleic acids, we split the RRE-RNA sequenceand connected to the complementary oligonucleotide fortarget nucleic acids. When each two oligonucleotideshybridized to an adjacent region of target nucleic acidscorrectly, a Rev-peptide binding site was reformed on thehybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase uponbinding to the hybridized complex in cell lysate. Theseresults suggest that the recombinant protein probe isavailable for specific nucleic acid detection.
-helical and elongatedconformation upon binding to Rev response element RNA(RRE-RNA) and Rev-aptamer, respectively. We confirmedthat YRGnC-11ad could specifically bind to RRE-RNA andRev-aptamer in cell lysate, and fluorescent resonanceenergy transfer (FRET) signal was changed upon bindingfollowing the conformational change of Rev-peptide. Toutilize this FRET signal change toward the detection ofspecific nucleic acids, we split the RRE-RNA sequenceand connected to the complementary oligonucleotide fortarget nucleic acids. When each two oligonucleotideshybridized to an adjacent region of target nucleic acidscorrectly, a Rev-peptide binding site was reformed on thehybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase uponbinding to the hybridized complex in cell lysate. Theseresults suggest that the recombinant protein probe isavailable for specific nucleic acid detection.