Romaniuk E, McLaughlin L W, Neilson T, Romaniuk P J
Eur J Biochem. 1982 Jul;125(3):639-43. doi: 10.1111/j.1432-1033.1982.tb06730.x.
In order to make efficient use of T4 RNA ligase in a program involving chemical-enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High-performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5')pp(5')Cp or A(5')pp(5')UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Conversely, examination of the four nucleoside 3',5'-bisphosphate donors pUp, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.
为了在涉及化学酶促寡核糖核苷酸合成的程序中有效利用T4 RNA连接酶，已对受体寡聚物的序列效应进行了优化。研究了供体分子对胞嘧啶磷酸二酯（pCp）和对尿嘧啶三磷酸胞苷（pUpUpUpCp）与一系列三核苷二磷酸ApApN、CpNpA和NpCpA（其中N = U、C、A或G）的反应。采用高效液相色谱法同时分析所有底物、中间体和产物。对于所有测试的三核苷二磷酸，均观察到大量中间体A(5')pp(5')Cp或A(5')pp(5')UpUpUpCp。这表明连接程度取决于受体分子的序列，而不取决于供体的腺苷酸化。相反，用共同受体ApApU对四种核苷3',5'-二磷酸供体pUp、pCp、pAp和pGp进行的研究表明，在连接效果较差的情况下，几乎不会形成中间体腺苷酸化供体。
