Amide-Modified RNA: Using Protein Backbone to Modulate Function of Short Interfering RNAs.
Amide-Modified RNA: Using Protein Backbone to Modulate Function of Short Interfering RNAs.
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DOI：
10.1021/acs.accounts.0c00249
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发表时间：
2020-09-15
影响因子：
18.3
通讯作者：
Rozners E
中科院分区：
文献类型：
--
作者：
Kotikam V;Rozners E
控制基因表达的基于RNA的技术，如RNA干扰（RNAi）和CRISPR-Cas9，已成为分子生物学和基因组学中的强大工具。RNAi和CRISPR-Cas9也可能成为新的治疗方法，这一令人兴奋的潜力重新激发了人们对化学修饰RNA以改善其体内应用特性的兴趣。化学修饰可以改善酶稳定性、体内递送、细胞摄取和序列特异性;以及最小化短干扰RNA（siRNA）和CRISPR相关RNA的脱靶活性。虽然已经出现了许多改善酶降解稳定性的良好解决方案，但后者功能特性的优化仍然具有挑战性。在这个帐户中，我们讨论了合成，结构和生物活性的新型非离子类似物的RNA，有磷酸二酯骨架取代酰胺键（AM 1）。我们的长期目标是利用酰胺骨架来提高siRNA和其他功能性RNA的稳定性和特异性。我们在这一领域的工作的动机是早期发现，非离子骨架修饰，包括AM 1，不干扰RNA双链体的整体结构或热稳定性。我们假设，AM 1骨架修饰的负电荷减少和疏水性质可能有助于通过增强细胞摄取来优化功能应用，并可能抑制siRNA的不必要的脱靶效应。NMR和X射线晶体学研究表明，AM 1是RNA中磷酸二酯键的极好模拟物。酰胺键引起的局部构象变化很容易通过RNA构象的微小调整来适应。此外，酰胺羰基呈现类似于非桥接P-O键之一的取向，这可以通过保持氢键相互作用来实现酰胺/磷酸酯模拟。与RNase H复合的短酰胺修饰的DNA-RNA杂合体的晶体结构表明，酰胺N-H也可以作为氢键供体来稳定RNA-蛋白质相互作用;这是磷酸基团不可用的相互作用模式。功能测定确定酰胺在siRNA的两条链中的内部位置处耐受良好。令人惊讶的是，与未修饰的siRNA相比，引导链中间和过客链5′端的酰胺修饰增加了RNAi活性。最重要的是，过客链的第一和第二核苷之间的酰胺键完全消除了其不期望的脱靶活性，同时增强了期望的RNAi活性。这些结果表明，RNAi可能比迄今为止尝试的化学方法更耐受siRNA的实质性修饰。这些发现也是重要和及时的，因为它们表明酰胺修饰可能会降低siRNA的脱靶活性，这仍然是RNAi临床应用的重要障碍。总之，我们的工作表明，酰胺键优化RNA的生物学和药理学特性的潜力未被充分认识。预期在RNA中扩大使用酰胺键以增强CRISPR和其他需要非编码RNA的化学稳定的功能模拟物的技术。
RNA-based technologies to control gene expression, such as, RNA interference (RNAi) and CRISPR-Cas9 have become powerful tools in molecular biology and genomics. The exciting potential that RNAi and CRISPR-Cas9 may also become new therapeutic approaches has reinvigorated interest in chemically modifying RNA to improve its properties for in vivo applications. Chemical modifications can improve enzymatic stability, in vivo delivery, cellular uptake, and sequence specificity; as well as minimize off-target activity of short interfering RNAs (siRNAs) and CRISPR associated RNAs. While numerous good solutions for improving stability towards enzymatic degradation have emerged, optimization of the latter functional properties remains challenging. In this Account, we discuss synthesis, structure, and biological activity of novel non-ionic analogues of RNA that have the phosphodiester backbone replaced by amide linkages (AM1). Our long-term goal is to use the amide backbone to improve the stability and specificity of siRNAs and other functional RNAs. Our work in this area was motivated by early discoveries that non-ionic backbone modifications, including AM1, did not disturb the overall structure or thermal stability of RNA duplexes. We hypothesized that the reduced negative charge and hydrophobic nature of the AM1 backbone modification might be useful in optimizing functional applications through enhanced cellular uptake, and might suppress unwanted off-target effects of siRNAs. NMR and X-ray crystallography studies showed that AM1 was an excellent mimic of phosphodiester linkages in RNA. The local conformational changes caused by the amide linkages were easily accommodated by small adjustments in RNA’s conformation. Further, the amide carbonyl group assumed an orientation that is similar to one of the non-bridging P-O bonds, which may enable amide/phosphate mimicry by conserving hydrogen bonding interactions. The crystal structure of a short amide-modified DNA-RNA hybrid in complex with RNase H indicated that the amide N-H could also act as an H-bond donor to stabilize RNA-protein interactions; which is an interaction mode not available to phosphate groups. Functional assays established that amides were well tolerated at internal positions in both strands of siRNAs. Surprisingly, amide modifications in the middle of the guide strand and at the 5′-end of the passenger strand increased RNAi activity compared to unmodified siRNA. Most importantly, an amide linkage between the first and second nucleosides of the passenger strand completely abolished its undesired off-target activity while enhancing the desired RNAi activity. These results suggest that RNAi may tolerate more substantial modifications of siRNAs than the chemistries tried so far. The findings are also important and timely because they demonstrate that amide modifications may reduce off-target activity of siRNAs, which remains an important roadblock for clinical use of RNAi. Taken together, our work suggests that amide linkages have underappreciated potential to optimize the biological and pharmacological properties of RNA. Expanded use of amide linkages in RNA to enhance CRISPR and other technology requiring chemically stable, functional mimics of non-coding RNAs is expected.
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DOI：
10.1002/anie.199402261
发表时间：
1994-02-01
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION IN ENGLISH
影响因子：
--
作者：
DEMESMAEKER, A;WALDNER, A;FREIER, SM
通讯作者：
FREIER, SM
影响因子：
4.3
作者：
Kotikam, Venubabu;Viel, Julien A.;Rozners, Eriks
通讯作者：
Rozners, Eriks
影响因子：
1.8
作者：
LEBRETON, J;WALDNER, A;DEMESMAEKER, A
通讯作者：
DEMESMAEKER, A
影响因子：
64.5
作者：
Elkayam E;Kuhn CD;Tocilj A;Haase AD;Greene EM;Hannon GJ;Joshua-Tor L
通讯作者：
Joshua-Tor L
影响因子：
46.9
作者：
Meade, Bryan R.;Gogoi, Khirud;Hamil, Alexander S.;Palm-Apergi, Caroline;van den Berg, Arjen;Hagopian, Jonathan C.;Springer, Aaron D.;Eguchi, Akiko;Kacsinta, Apollo D.;Dowdy, Connor F.;Presente, Asaf;Loenn, Peter;Kaulich, Manuel;Yoshioka, Naohisa;Gros, Edwige;Cui, Xian-Shu;Dowdy, Steven F.
通讯作者：
Dowdy, Steven F.