Plashkevych Oleksandr, Li Qing, Chattopadhyaya Jyoti
Chemical Biology Program, Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 581, SE-751 23 Uppsala, Sweden.
Mol Biosyst. 2017 May 2;13(5):921-938. doi: 10.1039/c6mb00762g.
A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pK 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg ion availability at the cleavage site causing sequence-specific hydrolysis of the phosphodiester bond of scissile phosphate. The enhancement of the total rate of cleavage of the complementary mRNA strand by up to 25%, presented in this work, provides opportunities to engineer a single modification site in appropriately substituted AONs to design an effective antisense strategy based on the nucleolytic stability of the AON strand versus RNase H capability to cleave the complementary RNA strand.
对36个单修饰的反义寡核苷酸- RNA异源双链体进行的详细动力学研究表明，在反义链（AON）中用锁核酸（LNA）或非对映体纯的碳环-LNA取代单个天然核苷酸，会导致核糖核酸酶H介导的互补mRNA链切割在5'-GpN-3'切割位点上发生2至5倍的位点依赖性调节，产生高达70%的RNA切割产物。实验是使用大肠杆菌的核糖核酸酶HI进行的。第二好的切割位点是5'-ApN-3'位点，其切割率高达23%，这取决于36个等序列互补AON中的取代位点。将修饰后的AON促进的RNA切割速率与天然AON的切割速率进行比较表明，修饰后序列特异性显著增强。显然，互补RNA链中相对较弱的5'-嘌呤（Pu）-嘧啶（Py）-3'堆积是优选的（产生约90%的总切割产物），这在核糖核酸酶H介导的RNA切割中起重要作用。有人提出核糖核酸酶H介导的RNA切割的一个合理机制有两个方面，由嘌呤糖苷配基的芳香性的平衡作用决定：第一，局部形成的9-鸟苷酸离子（pK 9.3，在pH 8时约18 - 20%的N1离子化）改变了切割磷酸周围相邻的糖-磷酸骨架，改变其糖N/S构象平衡，使其偏向S型，导致在20聚体互补mRNA中心周围的5'-GpN-3'位点优先切割。第二，5'-Pu-p-Py-3'堆积较弱的相邻强度促进了5'-GpN-3'和5'-ApN-3'的优先切割，提供了约90%的总产物，而天然AON的这一比例约为50%，这是由于cLNA/LNA取代基对相邻的5'-Pu-p-Py-3'位点的影响，既提供了局部空间灵活性又增加了水合作用。这有利于切割位点处水和水/镁离子的可及性，导致切割磷酸的磷酸二酯键发生序列特异性水解。本研究中互补mRNA链切割总速率提高了25%，这为在适当取代的AON中设计单个修饰位点提供了机会，以便基于AON链的核酸酶稳定性与核糖核酸酶H切割互补RNA链的能力来设计有效的反义策略。
