Martin Egli, Muthiah Manoharan
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接上篇文章《已获批准的寡核苷酸疗法的化学、结构和功能（一）》
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Physicochemical properties of oligonucleotides 寡核苷酸的物理化学性质
DNA and RNA are polyanions and form duplexes that are held together by H-bonding and stacking interactions between nucleobases. Stacking is ultimately more important for pairing stability than H-bonds; the latter of course provide specificity. The core of a nucleic acid duplex is essentially hydrophobic as water molecules are squeezed out upon base pairs collapsing on each other (hydrophobic effect) . The negative charges of phosphates are dotting the outside and the grooves created by the two strands winding around each other represent regions of varying negative electrostatic potential. Metal ions such as Mg2+, Ca2+, Na+ and K+ provide neutralizing charges, whereby both a diffuse counterion atmosphere (e.g. around DNA) and specific metal ion binding sites (e.g. Mg2+ in RNA) affect folding and stability.
DNA 和 RNA 是聚阴离子，形成的双链由核碱基之间的氢键和堆积相互作用保持在一起。堆积相互作用对于配对稳定性比氢键更为重要，而氢键当然提供了特异性。核酸双链的核心基本上是疏水的，因为水分子在碱基对相互叠合时被挤压出去（疏水效应）。磷酸的负电荷分布在外部，两条链缠绕在一起形成的沟槽代表了具有不同负电静电势的区域。镁离子（如 Mg2+）、钙离子（Ca2+）、钠离子（Na+）和钾离子（K+）提供了中和电荷，扩散的对离子环境（例如在 DNA 周围）和特定的金属离子结合位点（例如 RNA 中的 Mg2+）影响折叠和稳定性。
Cellular DNA is typically double-stranded and the pairing rules follow the Watson-Crick conventions, i.e. A:T and G:C. Conversely, RNA is single-stranded (tRNA, mRNA, rRNA), but folded RNAs exhibit extensive double-stranded regions. RNA duplexes are of the A-form with 11 base pairs per turn, a helical rise of <3 Å, and strongly inclined bases relative to the helical axis. RNA also displays tighter spacings between adjacent intra-strand phosphate groups compared to the physiological duplex type adopted by DNA, the so-called B-form. Base pairs in B-DNA are more or less normal to the direction of the helical axis, with an average rise of 3.4 Å and 10 base pairs per turn. DNA can also adopt the A-form and flip to left-handed Z-DNAas a result of torsional stress and dependent on sequence (CG-rich), ionic strengthand hydration level.
细胞内的 DNA 通常是双链的，配对规则遵循 Watson-Crick 规范，即 A:T 和 G:C。相反，RNA 是单链的（tRNA，mRNA，rRNA），但折叠的 RNA 展示了广泛的双链区域。RNA 双链属于 A 型结构，每个螺旋转数为 11 个碱基对，螺旋升高<3 Å，碱基相对于螺旋轴具有明显的倾斜角度。与 DNA 采用的生理双链结构（称为 B 型结构）相比，RNA 在链内磷酸基团之间显示出更紧密的间距。B-DNA 中的碱基对与螺旋轴的方向或多或少垂直，平均升高为 3.4 Å，每个螺旋转数为 10 个碱基对。DNA 也可以采用 A 型结构，并在扭曲应力和序列（CG 富集）、离子强度和水合水平的影响下翻转为左旋 Z-DNA。
The most significant difference between the backbones of DNA and RNA is the presence of the 2′-hydroxyl group in the ribose sugar of the latter. The 2′-hydroxyl group prefers an axial orientation and steric and stereoelectronic effects result in the preferred C3′-endo sugar conformation or pucker. In contrast, 2′-deoxyriboses in B-form DNA adopt a C2′-endo pucke. The 2′-OH group affects not just conformation but also stability and the water structure around the duplex. RNA duplexes are more stable than DNA duplexes of the same sequence and the gain is enthalpy-driven.
DNA 和 RNA 骨架之间最显著的区别是后者的核糖糖基中存在 2'-羟基。2'-羟基更倾向于轴向取向，并且由于空间位阻和立体电子效应，导致首选的 C3'-endo 糖构象或褶皱状态。相反，B 型 DNA 中的 2'-脱氧核糖糖采用 C2'-endo 褶皱构象。2'-羟基不仅影响构象，还影响双链周围的稳定性和水结构。相同序列的 RNA 双链比 DNA 双链更稳定，这种增益是由焓驱动的。
Both DNA and RNA can also form three- and four-stranded structures (triplexes and quadruplexes, respectively). Although DNA is capable of forming complex 3D structures in principle, as exemplified by a catalytic motif and nanostructures, it is clearly outdone by RNA as far as complex folds are concerned. As with sugar conformation and thermodynamic stability, RNA’s folding repertoire is aided by a multitude of interactions that involve the 2′-hydroxyl group, e.g. ribose zippers. Moreover, RNA base pairing is more versatile than the strict pairings in DNA, and G:U, A:C and pyrimidine:pyrimidine and purine:purine pairs as well as base triples and quadruples are commonplace. The 2′-hydroxyl group also plays a key role in RNA function and phenotype; thus, is acts as the nucleophile in self-cleaving reactions.
DNA 和 RNA 也可以形成三股和四股结构（分别称为三股结和四股结）。虽然 DNA 原则上可以形成复杂的三维结构，如催化基序和纳米结构，但在复杂折叠方面明显不及 RNA。与糖构象和热力学稳定性一样，RNA 的折叠能力受到众多相互作用的帮助，其中包括涉及 2'-羟基的核糖拉链。此外，RNA 的碱基配对比 DNA 更加灵活，常见的有 G:U、A:C 以及嘧啶:嘧啶和嘌呤:嘌呤的配对，还有碱基三联体和四联体。2'-羟基在 RNA 的功能和表型中也起着关键作用，因此在自剪切反应中充当亲核试剂。
More than 150 natural chemical modifications of RNA have been identified, among them 2′-O-methylation and complex decorations of nucleobases. It is now also recognized that DNA nucleobases are not limited to A, T, G and C, and that it is more appropriate to think of an expanded set of bases in the context of epigenetics. PS modifications have been established in the DNA of bacteria and archaea and they are exclusively of the Rp configuration. However, the PS modification does not appear to exist naturally in RNA. DNA PS modification is regulated by the Dnd gene cluster.
已经鉴定出 150 多种 RNA 的天然化学修饰，其中包括 2'-O-甲基化和核碱基的复杂修饰。现在人们也认识到 DNA 的碱基不仅限于 A、T、G 和 C，更适合在表观遗传学的背景下考虑扩展的碱基组。PS 修饰已在细菌和古菌的 DNA 中得到确认，且仅为Rp 构型。然而，在 RNA 中并不存在自然的 PS 修饰。DNA 的 PS 修饰受到Dnd基因簇的调控。
In line with the above-described differences in structure, stability and function between DNA and RNA, the physicochemical properties of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are quite distinct (Figure 4). A single-stranded DNA ASO has a molecular weight of typically 7 kDa and a 20mer would carry 19 negative charges. The single strand has a width of ca. 1 nM and is relatively flexible with the hydrophobic faces of bases partly stacked but otherwise accessible for protein interactions. In contrast, a typical siRNA duplex has a molecular weight of ca. 13 kDa and carries 40 negative charges. The duplex is more rigid than an ASO single strand and has a diameter of ca. 2 nM, whereby bases are tucked away in the core with virtually no hydrophobic faces exposed except for the 2-residue overhangs at the 3′-ends. These different physical/chemical properties result in marked differences in pharmacokinetic profiles. They also lead to very different chemical modification strategies, although phosphorothioates and 2′-functionality are of general importance for modification.
根据上述所描述的 DNA 和 RNA 之间的结构、稳定性和功能差异，反义寡核苷酸（ASO）和小干扰 RNA（siRNA）的物理化学性质是相当不同的（图 4）。单链 DNA ASO 的分子量通常为 7 kDa，20 个碱基组成的 20mer 带有 19 个负电荷。单链的宽度约为 1 纳米，相对灵活，碱基的疏水面部分堆叠，但也暴露于蛋白质相互作用。相比之下，典型的 siRNA 双链分子量约为 13 kDa，带有 40 个负电荷。双链比 ASO 单链更为刚性，直径约为 2 纳米，碱基被紧密包裹在核心区域，除了 3'端的 2 个残基外几乎没有暴露疏水面。这些不同的物理/化学性质导致药代动力学特性上的显著差异。它们还导致非常不同的化学修饰策略，尽管磷酸硫酯和 2'-功能性修饰是普遍重要的修饰方式。
图 4.
Antisense modes of action 反义作用模式
Rational or structure-based drug design requires knowledge of the 3D-structure of the target, typically an enzyme or a receptor. Screening of a compound library is used to identify initial leads and provides the starting point of an optimization via structure-activity relationships (SARs) that ideally results in a tightly binding candidate molecule for further in vitro and in vivo tests. But the path to a small-molecule drug is arduous, time-consuming and costly, and more often than not fails during the preclinical phase of the evaluation or later during the various phases of a clinical trial. The major attraction of the antisense strategy is that inhibition at the level of gene expression only requires knowledge of the targeted DNA or mRNA sequence. A 17mer oligonucleotide sequence is expected to occur only once in the human genome and the antisense approach therefore promises to be more efficient than the search for an enzyme inhibitor. Thus, therapeutic intervention at the nucleic acid level offers a rational path to drug discovery, whereby high specificity is afforded by Watson–Crick H-bonding interactions between ASO and target RNA.
合理的或基于结构的药物设计需要对目标的三维结构有所了解，通常是酶或受体。通过化合物库的筛选，可以识别出最初的引物，并通过结构活性关系（SARs）进行优化，以获得更紧密结合的候选分子，以进一步进行体外和体内测试。然而，小分子药物的开发过程艰难、耗时且昂贵，往往在临床前评估阶段或临床试验的各个阶段中失败。反义策略的主要吸引力在于，在基因表达水平上的抑制仅需要对目标 DNA 或 mRNA 序列有所了解。人类基因组中预计只会出现一次的 17 碱基寡核苷酸序列，因此反义方法比寻找酶抑制剂更有效。因此，在核酸水平上进行治疗干预为药物发现提供了一条理性的路径，其中通过 ASO 和靶标 RNA 之间的 Watson-Crick H 键相互作用实现高度特异性。
Although knowledge of the target sequence readily enables the design of an ASO, it is important to bear in mind that RNA molecules don’t exist as random coils. Because RNAs adopt intricate folds and are commonly decorated by proteins, many regions along the target sequence may be inaccessible to an ASO. Although efforts were made to predict sites on an RNA that allow ASO binding, an oligonucleotide screen—basically covering a large portion of the entire transcript with individual ASOs, e.g. 5′-UTR and 3′-UTR (Figure 5)—to identify the most active oligo in cell culture offers a more viable approach. In a very recent example of an oligo screen, Alnylam researchers synthesized 350 siRNAs against all available RNA genomes of SARS-CoV and SARS-CoV-2.
尽管对目标序列的了解可便利地实现反义寡核苷酸（ASO）的设计，但需要注意的是 RNA 分子并不以随机卷曲形式存在。由于 RNA 能够形成复杂的结构并常常与蛋白质相互作用，目标序列中的许多区域可能对 ASO 不可及。尽管已经努力预测 RNA 上允许 ASO 结合的位点，但通过对细胞培养中的每个 ASO 进行全面筛选（例如涵盖整个转录本的 5'-UTR 和 3'-UTR 区域，图 5），可以获得更可行的方法。在最近的一个 ASO 筛选示例中，Alnylam 的研究人员合成了 350 个针对 SARS-CoV 和 SARS-CoV-2 的所有可用 RNA 基因的 siRNA。
图 5.
For the further discussion of oligonucleotide therapeutics, it is helpful to properly define an ASO. It is a single stranded oligonucleotide that pairs with a complementary region on an RNA via Watson–Crick H-bonds and then acts, for example, via a steric block, splice site shifting or RNase-H mediated RNA degradation mechanism (Figure 5). On one hand, this contrasts with the antigene approach that targets DNA and functions via triplex formation, single-stranded decoys, and aptamers directed at DNA, RNA or proteins. On the other hand, double-stranded RNA molecules (siRNAs, ribozymes, and synthetic oligo metallonucleases all function via the antisense mechanism as well, although they are not typically referred to as ASOs.
为了进一步讨论寡核苷酸治疗药物，正确定义 ASO 是很有帮助的。ASO 是一种单链寡核苷酸，通过 Watson-Crick H 键与 RNA 上的互补区域配对，然后通过立体阻断、剪接位点移位或 RNase-H 介导的 RNA 降解机制等方式发挥作用（图 5）。一方面，这与靶向 DNA 并通过三链形成、单链诱饵和针对 DNA、RNA 或蛋白质的适配体等机制发挥作用的抗基因方法形成对比。另一方面，双链 RNA 分子（siRNA、核酶和合成寡核苷酸金属核酸酶）也通过反义机制发挥作用，尽管它们通常不被称为 ASO。
Perhaps categorizing antisense mechanisms is more useful than focusing on definitions that concern the oligonucleotides themselves. Thus, one can distinguish between mechanisms that involve binding and modulation of function without resulting in degradation of the target and others that promote cleavage of the targeted sequence. The former includes translation arrest, inhibition of translation initiation, inhibition of exon inclusion, promotion of exon inclusion, RNA antagonists (antagomirs), RNA agonists and disruption of RNA structure. Mechanisms that result in degradation of the targeted RNA can involve RNase H (elicited by an ASO), Argonaute2 (Ago2; RNAi), microRNA (miRNA; P-body and Ago proteins), U1 RNA adaptors and various catalytic motifs as well as chemically facilitated nucleases. Both RNase H and Ago2 are endogenous factors that play roles in natural pathways. A key difference between the actions of the two nucleases is that an ASO is bound to its RNA target prior to RNase H docking and cleavage. Conversely, the guide or antisense siRNA forms a complex with Ago2 before the enzyme interacts with the target.
也许将反义机制进行分类比关注于寡核苷酸本身的定义更有用。因此，可以区分涉及结合和调控功能而不导致目标降解的机制，以及促进目标序列的切割的机制。前者包括翻译抑制、翻译起始抑制、外显子包含的抑制、外显子包含的促进、RNA 拮抗物（antagomirs）、RNA 激动物和 RNA 结构破坏。导致目标 RNA 降解的机制可能涉及 RNase H（由 ASO 引发）、Argonaute2（Ago2；RNA 干扰）、microRNA（miRNA；P 体和 Ago 蛋白）以及 U1 RNA 适配体、各种催化基序和化学辅助核酸酶。RNase H 和 Ago2 都是在自然途径中发挥作用的内源因子。两个核酸酶之间的关键区别在于，在 RNase H 结合和切割之前，ASO 已经与其 RNA 靶点结合。相反，引导或反义 siRNA 在酶与靶点相互作用之前与 Ago2 形成复合物。
In this context, it is interesting to consider the evolution of our understanding of nucleic acid hybridization. As recounted by Alexander Rich, when he and David Davies demonstrated the spontaneous formation of a duplex by poly-A and poly-U, the observation was widely greeted with skepticism as many believed that a double helix could only be ‘made’ by an enzyme. The question therefore arises whether there are cellular factors that promote the hybridization of an ASO to its target? Indeed, it was reported that RNA binding proteins such as hnRNP A1 and YB1 significantly facilitate the hybridization of an oligonucleotide to RNA.
在这个背景下，考虑到我们对核酸杂交的理解是如何发展的是很有意思的。正如亚历山大·里奇（Alexander Rich）回忆的那样，当他和大卫·戴维斯（David Davies）展示了聚 A 和聚 U 之间自发形成双链的现象时，这一观察结果受到了广泛的怀疑，因为许多人认为双螺旋结构只能由酶“制造”。因此，问题出现了：是否存在促进 ASO 与其靶点杂交的细胞因子？事实上，有报道称 RNA 结合蛋白如 hnRNP A1 和 YB1 显著促进寡核苷酸与 RNA 的杂交。
Finally, in terms of a classification of antisense mechanisms and ASOs, it is important to distinguish cellular localizations, i.e. nucleus vs. cytoplasm . Thus, ASOs that modulate splicing, for example, by exon inclusion (SPINRAZA) or exon skipping (EXONDYS 51) act in the nucleus, whereas ASOs eliciting RNase H can operate either in the nucleus or in the cytoplasm. Subcellular localization of an ASO can affect its potency and the particular chemical makeup of an ASO impinges on its intracellular distribution.
最后，在对反义机制和 ASO 进行分类时，区分细胞定位，即细胞核与细胞质，是非常重要的。因此，调控剪接的 ASO（例如 SPINRAZA 通过外显子包含或 EXONDYS 51 通过外显子跳跃）作用于细胞核，而引发 RNase H 的 ASO 可以在细胞核或细胞质中发挥作用。ASO 的亚细胞定位可以影响其效力，而 ASO 的化学成分也会影响其细胞内分布。
The last point about an ASO’s chemistry having an effect on biodistribution is really an understatement as the chemical composition of an oligo affects pairing stability, metabolic stability, protein binding and toxicities caused by binding to Toll-like receptors, altered coagulation, immune cell and/or complement activation, off-target effects, pharmacokinetics and pharmacodynamics. Native DNA and RNA oligonucleotides are degraded rapidly in cells as they are unable to dodge attack by a host of exo- and endonucleases. RNA and RNA-like analogs exhibit higher pairing stability opposite RNA targets than DNA or DNA-like analogs. However, despite RNase H binding to both DNA and RNA duplexes, neither elicits enzyme action as the endonuclease recognizes a hybrid duplex in which the RNA is in the A-form and the DNA is in a B-like conformation.
关于 ASO 的化学组成对生物分布产生影响的观点确实是一种低调的陈述，因为寡核苷酸的化学组成会影响配对稳定性、代谢稳定性、蛋白结合以及与 Toll 样受体结合引起的毒性、凝血功能改变、免疫细胞和/或补体激活、非靶效应、药代动力学和药效动力学。原生 DNA 和 RNA 寡核苷酸在细胞内很快被外切酶和内切酶降解，无法抵御这些酶的攻击。RNA 和类似 RNA 的模拟物与 RNA 靶点形成的配对稳定性较 DNA 或类似 DNA 的模拟物更高。然而，尽管 RNase H 能够结合 DNA 和 RNA 双链，但它们都不能引发酶的作用，因为内切酶只识别呈 A 形态的 RNA、呈 B 形态的 DNA 杂交双链。
Uniform locked nucleic acids (LNAs) exhibit very high binding affinity for both DNA and RNA , but they also produce increased liver toxicity. Perhaps unexpectedly, uncharged peptide nucleic acids (PNAs) do not easily cross cell membranes, although the solubility can be increased with amino acids carrying charges in the PNA backbone. And, unlike PS-DNAs, PNA, PMO and regular phosphodiester-backbone oligonucleotides exhibit limited binding to plasma proteins, causing them to be rapidly excreted in the urine. Interestingly, the stabilin class of scavenger receptors has been shown to bind to PS-ASOs and aid in their cellular internalization. All these tidbits aim to illustrate that making drugs out of oligonucleotides is not easy and that modification chemistry was and remains crucial for overcoming numerous challenges on the way to nucleic acid therapeutics.
均一锁核酸（LNA）对 DNA 和 RNA 具有非常高的结合亲和力，但也会导致肝脏毒性增加。或许出人意料的是，尽管通过在 PNA 骨架中引入带电氨基酸可以增加其溶解性，但无电荷的肽核酸（PNA）仍不容易穿过细胞膜。与 PS-DNA 不同，PNA、PMO 和普通磷酸二酯骨架寡核苷酸与血浆蛋白的结合有限，导致它们在尿液中迅速排出。有趣的是，已经证明清道夫受体的稳定蛋白类可以与 PS-ASO 结合并有助于其细胞内摄取。所有这些细节都旨在说明将寡核苷酸转化为药物并非易事，而改性化学在克服通往核酸治疗的众多挑战中至关重要。
