Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.
Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.
复制标题
RNA 内环中连续 A.C、C.C、G.G、U.C 和 U.U 错配的稳定性：稳定氢键 U.U 和 C.C. 的证据
DOI：
10.1021/bi00247a021
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Turner,DH
中科院分区：
文献类型：
--
作者：
SantaLuciaJr,J;Kierzek,R;Turner,DH
Materials and MethodsRNA Synthesis and Purification. Oligoribonucleotides were synthesized on solid support with a modification of the method of Usman et al.(1987) with the 2'-hydroxyl protected as the ferf-butyl dimethylsilyl ether. Upon completion of the coupling reactions, the phosphate methyl groups were removed by treatment with thiophenol. The oligomer was removed from solid support, and base blocking groups were removed by treatment with concentrated ammonia in ethanol (3: 1 v/v) at 50 C overnight. The silyl protection was removed by treatment with 1.0 M triethylammonium hydrogen fluoride (50 equiv) in pyridine at 50 C for 48 h. The crude reaction mixture was then dried and partitioned between water and diethyl ether. The water layer was passed over a Sep-pak C-18 cartridge (Waters) to desalt. The oligomer was further pu-rified by HPLC on a PRP-1 semipreparative column (Hamilton) with a gradient of 0-50% acetonitrile buffered with 10 mM ammonium acetate, pH 7 (Ikuta et al., 1984). Dimethoxytrityl was removed by treating with 80% acetic acid for 30 min. Oligomers were desalted and further purified with a Sep-pak C-18 cartridge. Purities were checked by analytical C-8 HPLC (Beckman) and were greater than 95%. Melting Curves. The buffer for thermodynamic studies was 1.0 M NaCl, 10 mM sodium cacodylate, and 0.5 mM Na2E-DTA, pH 7. Solutions at pH 5.5 were 1.0 M NaCl, 50 mM MES, and 0.5 mM Na2EDTA. Single-strand extinction coefficients were calculated from extinction coefficients for dinucleoside monophosphates and nucleotides, as described previously (Borer, 1975; Richards, 1975). In units of 104 M" 1 cm" 1, the calculated extinction coefficients at 280 nm are as follows: rCGCCAGCG, 3.94; rCGCCCGCG, 4.00; rCGCCUGCG, 3.85; rCGCUCGCG, 3.77; rCGCUUGCG, 3.66; rGGCACGCC, 4.02; rUGCGGGCA, 3.82. Strand concentrations were determined from the high-temperature absorbance at 280 nm. Absorbance vs temperature melting curves were measured at 280 nm with a heating rate of 1.0 C min" 1 on a Gilford 250 spectrophotometer as described previously (Freier et al., 1983; Petersheim & Turner, 1983). Data Analysis. Absorbance vs temperature profiles were fit to a two-state model with sloping base lines by using a nonlinear least-squares program (Petersheim & Turner, 1983; Freier et al., 1983). Thermodynamic parameters for duplex formation were obtained by two methods:(1) enthalpy and
