TY - JOUR
T1 - Efficient synthesis of 13C,15N-labeled RNA containing the cap structure m7GpppA
AU - Matsuo, Hiroshi
AU - Moriguchi, Tomohisa
AU - Takagi, Toshimitsu
AU - Kusakabe, Takahiro
AU - Buratowski, Stephen
AU - Sekine, Mitsuo
AU - Kyogoku, Yoshimasa
AU - Wagner, Gerhard
PY - 2000/3/22
Y1 - 2000/3/22
N2 - Studying the mechanism by which the cap structure performs its numerous biological roles in mRNA metabolism by NMR spectroscopy requires the large- scale production of isotopically-labeled capped RNA. We present an efficient method for production of short RNA containing the cap structure m7GpppA, by combining chemical synthesis with enzymatic synthesis using a DNA primase. This method was employed to synthesize three capped RNA molecules, m7Gppp([13C,15N])A, m7Gppp([13C, 15N])ACC, and m7GpppA([13C,15N])C([13C,15N])C, that contain selective 13C,15N- labeled adenosine or cytosine nucleotides. This selective labeling technique enabled us to obtain the chemical shift assignments of these oligonucleotides in complex with the eukaryotic translation initiation factor 4E (eIF4E). Furthermore, we were able to observed intermolecular NOE interactions between the RNA and protein.
AB - Studying the mechanism by which the cap structure performs its numerous biological roles in mRNA metabolism by NMR spectroscopy requires the large- scale production of isotopically-labeled capped RNA. We present an efficient method for production of short RNA containing the cap structure m7GpppA, by combining chemical synthesis with enzymatic synthesis using a DNA primase. This method was employed to synthesize three capped RNA molecules, m7Gppp([13C,15N])A, m7Gppp([13C, 15N])ACC, and m7GpppA([13C,15N])C([13C,15N])C, that contain selective 13C,15N- labeled adenosine or cytosine nucleotides. This selective labeling technique enabled us to obtain the chemical shift assignments of these oligonucleotides in complex with the eukaryotic translation initiation factor 4E (eIF4E). Furthermore, we were able to observed intermolecular NOE interactions between the RNA and protein.
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U2 - 10.1021/ja9926820
DO - 10.1021/ja9926820
M3 - Article
AN - SCOPUS:0034701261
SN - 0002-7863
VL - 122
SP - 2417
EP - 2421
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 11
ER -