Department of Chemistry & Biochemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, Ohio 43210, United States.
Biochemistry. 2022 Jan 18;61(2):85-91. doi: 10.1021/acs.biochem.1c00693. Epub 2021 Dec 26.
General design principles for recognition at noncanonical interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust recognition platform that can be used to define structure-function relationships in synthetic triplex formation. To this end, a set of minimal ( < 1 kD) bPNA variants was synthesized to probe the impact of amino acid secondary structural propensity, stereochemistry, and backbone cyclization on hybridization with short, unstructured T-rich DNA and U-rich RNAs. Thermodynamic parameters extracted from optical melting analyses of bPNA variant hybrids indicated that there are two bPNA backbone modifications that significantly improve hybridization: alternating (d, l) configuration in open-chain dipeptides and dipeptide cyclization to diketopiperazine. Further, binding to DNA is preferred over RNA for all bPNA variants. Thymine-uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for 71% of ΔΔ for open-chain bPNAs but only 40% of ΔΔ for diketopiperazine bPNA, suggesting a greater sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic base presentation (as found in homochiral diketopiperazines and d, l peptides), expansion of base stacking, and further investigation of bPNA backbone preorganization.
非canonical 界面的 DNA 和 RNA 识别的一般设计原则仍然难以捉摸。双面肽核酸 (bPNA) 与寡聚 T/U DNA 和 RNA 的三聚体杂交是一种强大的识别平台，可用于定义合成三聚体形成中的结构-功能关系。为此，合成了一组最小的 (<1 kD) bPNA 变体，以探究氨基酸二级结构倾向、立体化学和骨架环化对与短、无规 T 富 DNA 和 U 富 RNA 杂交的影响。从 bPNA 变体杂交体的光学熔融分析中提取的热力学参数表明，有两种 bPNA 骨架修饰可以显著提高杂交效率：开链二肽中的交替 (d, l) 构型和二肽环化形成二酮哌嗪。此外，所有 bPNA 变体都优先与 DNA 结合，而不是与 RNA 结合。DNA 底物中的胸腺嘧啶-尿嘧啶取代表明，胸腺嘧啶的甲基基团占开链 bPNA 的 ΔΔ的 71%，但仅占二酮哌嗪 bPNA 的 ΔΔ的 40%，这表明对 RNA 构象的敏感性更高，并且在环状 bPNA 中更优化的堆积。这些数据共同揭示了在 bPNA 的手性中心、骨架构象、碱基三联体的堆积效应以及核酸底物本身调整三聚体杂交的关键点。增强 bPNA 对 DNA 和 RNA 底物的靶向的结构蓝图包括手性二酮哌嗪和 d, l 肽中发现的同手性碱基呈现、碱基堆积的扩展以及对 bPNA 骨架预组织的进一步研究。
