Endoh Tamaki, Funabashi Hisakage, Mie Masayasu, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Yokohama, 226-8501, Japan.
Anal Chem. 2005 Jul 15;77(14):4308-14. doi: 10.1021/ac048491j.
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.
检测特定核酸对于理解细胞机制和基因调控功能至关重要。在此，我们展示了一种利用重组蛋白和寡核苷酸检测特定核酸的新方法。构建了一种重组蛋白YRGnC - 11ad，其在增强型黄色荧光蛋白（EYFP）和增强型青色荧光蛋白（ECFP）之间含有Rev肽，并在HeLa细胞中表达。Rev肽对应于HIV - 1 Rev蛋白的第34 - 50个氨基酸，在溶液中呈无序结构，但分别与Rev反应元件RNA（RRE - RNA）和Rev适配体结合时形成α - 螺旋和伸长构象。我们证实YRGnC - 11ad能在细胞裂解物中特异性结合RRE - RNA和Rev适配体，并且随着Rev肽构象变化，结合后荧光共振能量转移（FRET）信号发生改变。为利用这种FRET信号变化来检测特定核酸，我们将RRE - RNA序列拆分，并连接到用于靶核酸的互补寡核苷酸上。当每两个寡核苷酸正确杂交到靶核酸的相邻区域时，杂交复合物上会重新形成Rev肽结合位点。并且我们能够证实在细胞裂解物中，YRGnC - 11ad重组蛋白与杂交复合物结合后FRET增加。这些结果表明重组蛋白探针可用于特定核酸检测。
