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Cho, Bong Rae,Pyun, Sang Yong Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.4
Elimination reactions of $(E)-2,4-(NO_2)_2C_6H_2CH=NOC_6H_3-2-X-4-NO_2$ (1a-e) promoted by $R_3N/R_3NH^+$ in 70 mol % MeCN(aq) have been studied kinetically. The reactions are second-order and exhibit Br$\ddot{o}$nsted ${\beta}$ = 0.80-0.84 and ${\mid}{\beta}_{lg}{\mid}$ = 0.39-0.42, respectively. For all leaving groups and bases employed in this study, the ${\beta}$ and ${\mid}{\beta}_{lg}{\mid}$ values remained almost the same. The results can be described by a negligible $p_{xy}$ interaction coefficient, $p_{xy}={\partial}{\beta}/pK_{lg}={\partial}{\beta}_{lg}/pK_{BH}{\approx}0$, which describes the interaction between the base catalyst and the leaving group. The negligible pxy interaction coefficient is consistent with the $(E1cb)_{irr}$ mechanism. Change of the base-solvent system from $R_3N$/MeCN to $R_3N/R_3NH^+$-70 mol % MeCN(aq) changed the reaction mechanism from E2 to $(E1cb)_{irr}$. Noteworthy was the relative insensitivity of the transition state structure to the reaction mechanism change.
Isolation of New Self-Cleaving Ribozymes with in vitro Selection
Cho, Bong-Rae,Lee, Young-Hoon Korean Chemical Society 2005 Bulletin of the Korean Chemical Society Vol.26 No.12
In vitro selection was used to isolate $Mg^{2+}$-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli $tRNA^{Phe}$. After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of $Mg^{2+}$ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM $Mg^{2+}$ but not in 10 mM $Mg^{2+}$. The cleavage sites of the ribozymes are located at +3 and +4 of $tRNA^{Phe}$, compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and $tRNA^{Leu}$ showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with $tRNA^{Phe}$. Our results suggest that the selected ribozyme is not structural-specific for tRNA.
In Vitro Selection of Hammerhead Ribozymes with Optimized Stems Ⅰand Ⅲ
Cho, Bong Rae,Lee, Young Hoon,Park, In Won,Kim, Ha Dong,Kim, Se Mi,AHN, JEONG KEUN,Sim, So Yeong 생화학분자생물학회 1999 BMB Reports Vol.31 No.2
A pool of cis-acting hammerhead ribozymes randomized in their substrate recognition sequences was constructed. A variety of active cis-acting ribozymes which had various structures of stems Ⅰ and Ⅲ was selected from the pool by in vitro selection. The selected ribozymes were cloned and sequenced. The relationship between the cleavage efficiency and basepairing in stems Ⅰ and Ⅲ of the selected ribozymes was investigated. The ribozymes with the smaller difference in folding energies between the active conformation and the stable but inactive conformation showed a tendency to have the better cleavage efficiency. The optimum length of stem Ⅰ was 5 or 6 bases while the longer stem Ⅲ, in general, appeared to be required for efficient cleavage. The specificity of the ribozyme reaction is discussed in terms of the length of stems Ⅰ and Ⅲ.