Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost.
Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost.
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DOI：
10.1021/acs.biochem.0c00685
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发表时间：
2020-12
期刊：
影响因子：
2.9
通讯作者：
P. Pallan;T. Lybrand;M. Schlegel;J. Harp;Hartmut Jahns;M. Manoharan;M. Egli
中科院分区：
文献类型：
--
作者：
P. Pallan;T. Lybrand;M. Schlegel;J. Harp;Hartmut Jahns;M. Manoharan;M. Egli
GNRA（N = A、C、G 或 U；R = A 或 G）四环是常见的 RNA 二级结构基序，其特征是磷酸盐堆叠在核碱基顶部。 rRNA 八叠球菌毒素/蓖麻毒素环 (SRL) 被 GApGA 封端，磷酸盐 p 堆积在 G 上。我们最近发现，区域特异性掺入单个二硫代磷酸盐 (PS2)，而不是单硫代磷酸盐 (PSO)，而不是 RNA 适体主链中的磷酸盐，可显着增加与其靶标的结合亲和力。在RNA：凝血酶复合物中，使结合紧密1000倍的关键在于PS2和苯丙氨酸环之间的边缘接触。在这里，我们研究了用 PS2 或 PSO 替换与鸟嘌呤面对面相互作用的 SRL 磷酸盐以保持稳定性的后果。我们发现，与母环相比，PS2·G 和 Rp-PSO·G 接触将修饰的 SRL 稳定到意想不到的水平：熔化温度 Tm 高达 6.3 °C，ΔΔG° 高达 -4.7 kcal/mol。晶体结构表明，尽管前者的范德华半径较大（S 为 1.80 Å，O 为 1.52 Å），但最接近的硫与鸟嘌呤的垂直距离平均仅比氧长 0.05 Å。更高的稳定性是基于焓的，并且通过中性甲基膦酸酯修饰评估的负电荷仅起次要作用。量子力学/分子力学计算支持硫占主导地位的有利色散吸引相互作用。除了 GNRA 之外，在新分类的 RNA 四环家族中也发现了磷酸盐和鸟嘌呤 (SRL) 或尿嘧啶（U 形转弯）之间的堆积相互作用。
GNRA (N = A, C, G, or U; R = A or G) tetraloops are common RNA secondary structural motifs and feature a phosphate stacked atop a nucleobase. The rRNA sarcin/ricin loop (SRL) is capped by GApGA, and the phosphate p stacks on G. We recently found that regiospecific incorporation of a single dithiophosphate (PS2) but not a monothiophosphate (PSO) instead of phosphate in the backbone of RNA aptamers dramatically increases the binding affinity for their targets. In the RNA:thrombin complex, the key contribution to the 1000-fold tighter binding stems from an edge-on contact between PS2 and a phenylalanine ring. Here we investigated the consequences of replacing the SRL phosphate engaged in a face-on interaction with guanine with either PS2 or PSO for stability. We found that PS2···G and Rp-PSO···G contacts stabilize modified SRLs compared to the parent loop to unexpected levels: up to 6.3 °C in melting temperature Tm and -4.7 kcal/mol in ΔΔG°. Crystal structures demonstrate that the vertical distance to guanine for the closest sulfur is just 0.05 Å longer on average compared to that of oxygen despite the larger van der Waals radius of the former (1.80 Å for S vs 1.52 Å for O). The higher stability is enthalpy-based, and the negative charge as assessed by a neutral methylphosphonate modification plays only a minor role. Quantum mechanical/molecular mechanical calculations are supportive of favorable dispersion attraction interactions by sulfur making the dominant contribution. A stacking interaction between phosphate and guanine (SRL) or uracil (U-turn) is also found in newly classified RNA tetraloop families besides GNRA.