研究报告: 基于遗传算法的计算机辅助策略优化孕酮适配体

目的 本研究致力于优化孕酮（progesterone，P4）适配体的亲和力和选择性。方法 基于遗传算法（genetic algorithm，GA）的计算机辅助优化策略（in silico maturation，ISM），进行了4轮GA操作（含交叉变异、单点突变和双点突变操作），构建了初始文库和G1、G2、G3代ssDNA作为新的候选适配体库，采用分子对接对候选适配体进行筛选和分析，并使用迭代策略不断优化适配体。此外，还提出了一种较为准确预测ssDNA三级结构的方法，首先使用Mfold预测二级结构，继而使用RNAComposer建立与ssDNA相对应的RNA三级结构，输出的PDB文件使用Discovery Studio将RNA修改为DNA，最后使用Molecular Operating Environment对结构进行能量最小化处理。结果 到G2代，在局部搜索空间对P4S-0进行优化，筛选出P4G1-14、P4G2-20、P4G1-6、P4G1-7和P4G2-14这5条适配体作为P4的最佳候选适配体。采用AuNPs比色法初步验证优化后适配体的亲和力，继而构建了基于适配体结构开关的荧光法测定适配体的解离常数（equilibrium dissociation constant，K_D），并以此方法对适配体的选择性（对双酚A、雌二醇、睾酮和皮质醇）进行了评估。结论 通过ISM优化后的适配体，对P4的亲和力较原适配体有了较大提升，仍保留着识别结构类似分子的选择性。

Review: Optimization of Progesterone Aptamers in silico Based on Genetic Algorithm

Objective The purpose of this study is to improve the affinity and selectivity of aptamers of progesterone (P4).Methods The computer-aided optimization strategy (in silico maturation, ISM) based on genetic algorithm (GA) was used. The 4 rounds of GA (including crossover mutation, single point mutation and double point mutation operations) were performed to construct the initial library and G1, G2 and G3 generation ssDNA oligonucleotides as new candidate aptamer libraries. The candidate aptamers were isolated and analyzed by molecular docking, and continuously optimized using an iterative strategy. In addition, a new strategy is proposed to predict the tertiary structure of ssDNA more accurately. Their secondary and tertiary structures were modeled by Mfold and RNA composer respectively. Output PDB files were modified from RNA to DNA in the Discovery Studio software. Finally, the structure is energy minimized by Molecular Operating Environment.Results In the second generation, P4S-0 sequence was optimized in the local search space, and P4G1-14, P4G2-20, P4G1-6, P4G1-7 and P4G2-14 were selected as the best aptamers for P4. AuNPs colorimetric method was applied to verify the affinity of the optimized aptamer. And then a fluorescence method based on the structure switch of the aptamer was constructed to determine the dissociation constant (K_D) of the aptamer, and the selectivity of the aptamer (for bisphenol A, estradiol, testosterone and cortisol) was evaluated.Conclusion The aptamer optimized by ISM has a greater affinity for P4 than the original aptamer, and still retains the selectivity for recognizing molecules with similar structure.