已获批准的寡核苷酸疗法的化学、结构和功能（四）

《已获批准的寡核苷酸疗法的化学、结构和功能（一）》
《已获批准的寡核苷酸疗法的化学、结构和功能（二）》
接上篇《已获批准的寡核苷酸疗法的化学、结构和功能（三）》

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SECOND-GENERATION MODIFICATIONS 第二代修饰

Conformational preorganization and gapmer approach 空间预组织和间隔核苷酸策略

The RNA 2′-hydroxyl group shifts the conformational equilibrium of the sugar ring to the so-called North or C3′-endo conformation. There is a linear correlation between the electronegativity of the 2′-substituent and the ratio between the North/South sugar conformations. Although C3′-endo is the pucker mode preferred by ribonucleotides, the sugar is by no means frozen and RNA nucleotides (e.g. in tRNA) adopt a Southern conformation on occasion. In 2′-OMe-RNA and 2′-F-RNA (Figure 7B) the gauche effect between 2′-substituent and O4′ is maintained and both exhibit a preference for the C3′-endo sugar conformation. These analogs paired opposite RNA increase the thermodynamic stability of the resulting duplex compared with native RNA. The methyl moiety in 2′-OMe-RNA points into the minor groove and the C3′–C2′–O2′–Me torsion angle is always in the antiperiplanar range. DNA sugars favor the South or C2′-endo conformation, but the conformational equilibrium can be shifted to the C3′-endo pucker by altering the gauche effect between O3′ and O4′. Thus, the 3′-methylene and N3′→P5′ phosphoramidate DNA analogs mimic RNA and prefer the C3′-endo sugar pucker.

RNA 的 2'-羟基团将糖环的构象平衡转移到所谓的"北"或 C3'-内型构象。2'-取代基的电负性与"北"/"南"糖构象比之间存在线性相关性。虽然 C3'-内型是核糖核苷酸首选的褶曲方式，但糖并不是冻结的，RNA 核苷酸（例如在 tRNA 中）偶尔也会采用"南"构象。在 2'-OMe-RNA 和 2'-F-RNA（图 7B）中，2'-取代基和 O4'之间的手性效应得到保持，并且两者都倾向于 C3'-内型糖构象。这些类似物与 RNA 相互配对时，与天然 RNA 相比，形成的双链具有更高的热力学稳定性。2'-OMe-RNA 中的甲基基团指向小凹槽，C3'-C2'-O2'-Me 扭转角始终在反位构型范围内。DNA 糖偏好于"南"或 C2'-内型构象，但可以通过改变 O3'和 O4'之间的手性效应将构象平衡转移到 C3'-内型褶曲。因此，3'-亚甲基和 N3'→P5'磷酸酰胺酯 DNA 类似物模拟 RNA，并倾向于 C3'-内型糖褶曲。

In chimeric RNA-DNA oligonucleotides, ribonucleotides can conformationally dominate the DNA portion to various degrees. We and others found in crystal structures that a single or a few ribonucleotides can flip a 10mer DNA duplex into the A-form. It is possible that this effect is influenced by lattice forces and dehydration as well as sequence, as the degree of conformational dominance appears to be different in solution. Also, in the crystal structure of the duplex [d(CGCGAA)-UU-d(CGCG)]2 with 2′-SMe-U (U), only underlined nucleotides adopt a C3′-endo sugar pucker. Neighboring 2′-deoxyadenosines retain a C2′-endo pucker and the RNA-like modified uridines in the center force just the 3′-adjacent cytidine into a North conformation. As a result of the sugar pucker switch from South to North and back to South due to the presence of the 2′-SMe substituents, the minor groove in the center of the duplex widens significantly relative to the parent DNA duplex. The EcoRI restriction endonuclease cleaves the all-DNA strand between G and A (bold font) in the recognition sequence 5′-GAATTC-3′/3′-CTTAAG-5′. However, when either one of the Ts or both are replaced by 2′-S*eMe-U endonuclease activity is completely abolished, thus demonstrating that nucleic acid-binding and/or processing proteins are sensitive to local changes in backbone geometry and duplex conformation.

在嵌合的 RNA-DNA 寡核苷酸中，核糖核苷酸可以以不同程度对 DNA 部分产生构象上的优势。我们和其他研究人员在晶体结构中发现，少量核糖核苷酸可以将一个 10 碱基的 DNA 双链转变为 A 形构象。这种效应可能受到晶格力和脱水以及序列的影响，因为在溶液中构象优势的程度似乎是不同的。此外，在[d(CGCGAA)-UU-d(CGCG)]2 的晶体结构中，使用 2'-SMe-U（U）替代的核苷酸仅有下划线下的核苷酸采用 C3'-内型糖褶曲。相邻的 2'-去氧腺苷保持 C2'-内型褶曲，而中间的类似 RNA 修饰的尿苷使得 3'-相邻的胞苷转为"北"构象。由于存在 2'-SMe 取代基，糖褶曲从"南"到"北"再到"南"的转变，使得双链中心的小凹槽相对于亲本 DNA 双链显著加宽。EcoRI 限制内切酶在识别序列 5'-GAATTC-3'/3'-CTTAAG-5'中的 G 和 A 之间（粗体字）切割全 DNA 链。然而，当一个或两个胸腺嘧啶被 2'-S*eMe-U 替代时，内切酶活性完全消失，从而证明核酸结合和/或处理蛋白对于背骨几何和双链构象的局部变化非常敏感。

The nucleotide sugar pucker is also a key determinant of the catalytic efficiency with which DNA polymerases (pols) incorporate dNTPs into the growing chain, whereby preferences vary widely among different pols. DNA primer insertion and extension assays using dATP analogs with methanocarba bicyclo[3.1.0]hexane sugar scaffolds that lock the pucker either in the North C2′-exo or South C3′-exo forms (N-MC-dATP and S-MC-dATP, respectively) identified distinct behaviors by so-called Y-family error bypass pols. Interestingly, given that they are DNA pols, human pol η, κ and ι all preferably or exclusively incorporate N-MC-dATP. In the steady-state kinetic analysis, the efficiency (kcat/Km) relative to incorporation of dATP was decreased 4-fold (pols η and κ) and increased 5-fold (pol ι). Neither pol κ nor pol ι were able to incorporate S-MC-dATP, but the former could at least extend from the N-MC-dA nucleotide. Conversely, pol η was able to also incorporate S-MC-dATP, albeit with an 80-fold decreased efficiency relative to dATP, and to extend from both the N- and S-MC-dA nucleotides. As expected, HIV reverse transcriptase preferred N-MC-dATP and the efficiency of incorporation was unchanged relative to that of dATP.

核苷酸糖褶皱也是决定 DNA 聚合酶（pol）催化效率的关键因素之一，这些酶将 dNTPs 插入到增长的链中，不同的 pol 在偏好方面存在广泛差异。使用将糖褶皱锁定在北 C2'-外型或南 C3'-外型形式（分别为 N-MC-dATP 和 S-MC-dATP）的甲基卡巴碳桥[3.1.0]己烷糖支架的 dATP 类似物进行 DNA 引物插入和延伸实验，发现所谓的 Y 家族错误旁路聚合酶表现出不同的行为。有趣的是，尽管它们是 DNA 聚合酶，但人类 pol η、κ和ι都更倾向于或专门插入 N-MC-dATP。在稳态动力学分析中，相对于 dATP 的插入效率（kcat/Km）下降了 4 倍（pol η和κ），增加了 5 倍（pol ι）。无论是 pol κ还是 pol ι都无法插入 S-MC-dATP，但前者至少能从 N-MC-dA 核苷酸延伸。相反，pol η也能够插入 S-MC-dATP，尽管相对于 dATP 而言效率降低了 80 倍，并且能够从 N-MC-dA 和 S-MC-dA 核苷酸进行延伸。正如预期的那样，HIV 逆转录酶更倾向于 N-MC-dATP，其插入效率相对于 dATP 没有变化。

The distinct behaviors of the DNA pols vis-à-vis nucleotides of opposite puckers were also seen with the B-family Dpo1 and Y-family Dpo4 pols from the archaeal hyperthermophile Sulfolobus solfataricus. Thus, the former inserts both the N- and S-MC-dATPs with relatively minor reductions in efficiency, whereas the latter only inserts N-MC-dATP. Dpo4 was then unable to extend; extensions by Dpo1 from the MC-dA nucleotides were inhibited in both cases relative to dA, whereby incorporation following S-MC-dA was slightly favored. The tolerance toward both fixed conformation nucleotide analogs exhibited by human pol η foreshadowed the enzyme's ability to incorporate ribonucleotide triphosphates relatively efficiently, act as a reverse transcriptase, accommodate RNA for strand extension, and bind to DNA–DNA, DNA–RNA and RNA–RNA duplexes with similar affinities.

在古细菌超热嗜好菌Sulfolobus solfataricus的 B 家族 Dpo1 和 Y 家族 Dpo4 聚合酶中，对于具有不同糖褶皱的核苷酸，也观察到了不同的行为。因此，前者插入 N-MC-dATP 和 S-MC-dATP 时的效率略有降低，而后者仅插入 N-MC-dATP。然后，Dpo4 无法进行延伸；Dpo1 在两种情况下从 MC-dA 核苷酸的延伸相对于 dA 受到抑制，而在插入 S-MC-dA 后稍微偏好于插入。人类 pol η对于固定构象的核苷酸类似物的耐受性预示了该酶相对高效地插入核糖核苷酸三磷酸盐，作为逆转录酶的功能，容纳 RNA 进行链延伸，并与 DNA-DNA、DNA-RNA 和 RNA-RNA 双链具有类似的亲和力结合。

RNase H also displays affinity for all three duplex types as well as for single strands, but it binds DNA–RNA hybrid and RNA duplexes ca. 60-fold more tightly than DNA duplexes and ca. 300-fold more tightly than single stranded nucleic acids. However, as indicated before the enzyme only processes the RNA strand opposite DNA, although it is noteworthy that RNase HI from the thermoacidophilic archaeon Sulfolobus tokodaii was reported to cleave RNA strands opposite either DNA or RNA. RNase H and Ago2—the former targeting RNA opposite antisense DNA, the latter RNA opposite antisense (guide) RNA—catalyze the same reaction, namely phosphodiester hydrolysis via a dual metal ion mechanism that results in formation of two strands, one with a free 3′-hydroxyl group and the other with a 5′-phosphate group (Figure 8A). The PIWI domain that harbors the RNA endonuclease activity in Ago2 also adopts an RNase H-type fold (Figure 8B).

RNase H 对三种双链结构以及单链结构都表现出亲和力，但它与 DNA-RNA 杂交和 RNA 双链结构的结合强度约为 DNA 双链结构的 60 倍，单链核酸的结合强度约为 300 倍。然而，正如之前所提到的，该酶只处理与 DNA 相对的 RNA 链，尽管值得注意的是，来自热酸性嗜好古菌Sulfolobus tokodaii的 RNase HI 据报道能够切割与 DNA 或 RNA 相对的 RNA 链。RNase H 和 Ago2（前者靶向反义 DNA 对位的 RNA，后者靶向反义（引导）RNA 对位的 RNA）催化相同的反应，即通过双金属离子机制进行磷酸二酯水解，形成两条链，一条具有自由的 3'-羟基，另一条具有 5'-磷酸基团（图 8A）。在 Ago2 中承载 RNA 内切酶活性的 PIWI 结构域也采用了 RNase H 类型的折叠结构（图 8B）。

图 8.

In the crystal structure of a bacterial RNase H bound to an RNA–DNA hybrid, the RNA strand is properly oriented at the active site with two Mg2+ ions coordinated by Asp and Glu residues and the scissile phosphate lined up to be attacked by the water nucleophile. Conversely, the complex of Ago2 with an siRNA duplex is not trapped in an active conformation, Mg2+ ions are absent, and the sense strand does not reach the active site (seed region complex). An overlay of the Ago2 Piwi domain and RNase H from the two complex structures demonstrates that the respective RNA strands exhibit different curvatures (Figure 8B) and exposes Ago2 loop regions that need to recoil for the scissile phosphate to move into the active site.

在细菌 RNase H 与 RNA-DNA 杂交体的晶体结构中，RNA 链在活性位点上以正确的方向定位，两个 Mg2+离子由 Asp 和 Glu 残基配位，并使被水亲核试剂攻击的切割磷酸盐排列整齐。相反，Ago2 与 siRNA 双链体的结构并未陷入活性构象，缺少 Mg2+离子，而正义链未达到活性位点（种子区结合）。将 Ago2 Piwi 结构域和 RNase H 的两个复合物结构进行重叠显示，可以看到各自的 RNA 链呈现不同的曲率（图 8B），并暴露出 Ago2 回路区域，这些区域需要收缩以使切割磷酸盐移动到活性位点。

RNase H probes the width of the minor groove of the duplex it docks onto, whereby that groove in an RNA-DNA substrate is narrower and wider than the corresponding grooves in RNA and DNA duplexes, respectively. An overlay of crystal structures of Bacillus halodurans RNase H bound to an RNA-DNA substrate duplex and a DNA–DNA inhibitor duplex of the same length shows that the groove widths and strand curvatures in the two duplexes differ distinctly (Figure 8C). RNase H interacts directly with several 2′-hydroxyl groups of the RNA strand and the lack of these contacts in the complex with a DNA duplex precludes proper recognition and of course processing, even if the 2′-OH groups are not directly involved in cleavage. The importance of the 2′-OH interactions for substrate recognition and action by RNase H is further demonstrated by the observation that the enzyme does not cleave a 2′-F-RNA strand opposite DNA. The absence of metal ions at the RNase H active site in the structure of the complex with the 2′-F-RNA–DNA duplex further highlights the key role played by 2′-hydroxyls in the proper functioning of the enzyme.

RNase H 探测其所结合的双链体的次沟宽度，在 RNA-DNA 底物中，该沟比 RNA 和 DNA 双链体的相应沟窄或宽。将 Bacillus halodurans RNase H 结合于 RNA-DNA 底物双链体和长度相同的 DNA-DNA 抑制剂双链体的晶体结构进行重叠，显示两个双链体的沟宽和链曲率明显不同（图 8C）。RNase H 直接与 RNA 链的几个 2'-羟基进行相互作用，而在与 DNA 双链体形成的复合物中缺乏这些接触，阻止了适当的识别和处理，即使 2'-羟基不直接参与切割。进一步证明了 2'-羟基与 RNase H 底物识别和作用的重要性，观察到该酶不会切割 2'-F-RNA 链与 DNA 相对应的序列。在与 2'-F-RNA-DNA 双链体形成的复合物的结构中，RNase H 活性位点缺乏金属离子进一步凸显了 2'-羟基在酶的正常功能中的关键作用。

Importantly, 2′-F-RNA as antisense oligo paired to a complementary RNA abrogates cleavage of the latter by RNase H, as do all ASOs that adopt an A-form geometry. Thus, a uniform 2′-O-modification strategy cannot be applied with ASOs that are intended to elicit RNase H and act via degradation of the RNA target. It also sheds light on the origins of the inability of RNase H to cleave RNA opposite a DNA containing 4′-thio-2′-deoxynucleotides. The gapmer ASO design provides a solution to this conundrum: the central PS-DNA window promotes cleavage by RNase H and 2′-O-modified wings afford stability. The central region is typically between six and ten nucleotides long and the PS modification ensures metabolic stability and favorable biodistribution compared to native DNA. Evidence has been presented that RNase H enzymes prefer particular sequences, and it turns out that RNase H is exquisitely sensitive to conformational changes induced by chemical modifications in the strand opposite RNA. Two of only a handful of ASO modifications that are tolerated by RNase H are arabinonucleic acid (ANA) and 2′-deoxy-2′-fluoro-arabinonucleic acid (FANA). FANA nucleotides in a DNA duplex background were found early on to adopt an Eastern O4′-endo pucker and both ANA and FANA appear capable of mimicking the DNA strand geometry in a hybrid duplex with RNA that is recognized and processed by RNase H.

2'-F-RNA 或采用 A 形态几何构型的 ASO，与互补的 RNA 形成配对时，会阻止 RNase H 对后者的切割，与所有采用 A 形态几何构型的 ASO 一样。因此，在旨在通过降解 RNA 靶点来引发 RNase H 作用的 ASO 中，无法采用统一的 2'-O-修饰策略。这也揭示了 RNase H 无法切割与含有 4'-硫基-2'-脱氧核苷酸的 DNA 相对的 RNA 的原因。gapmer ASO 设计提供了解决这个难题的方案：中央的 PS-DNA 窗口促进了 RNase H 的切割，而 2'-O-修饰的翼部分则提供了稳定性。中央区域通常由六到十个核苷酸组成，并且 PS 修饰确保了与天然 DNA 相比的代谢稳定性和有利的生物分布。已经提供了 RNase H 对特定序列的偏好的证据，事实证明 RNase H 对 RNA 对侧发生的化学修饰引起的构象变化非常敏感。RNase H 所容忍的 ASO 修饰中的两个例子是阿拉伯核酸（ANA）和 2'-脱氧-2'-氟阿拉伯核酸（FANA）。早期的研究发现，将 FANA 核苷酸嵌入 DNA 双链背景中可采用东方的 O4'-内环糖曲线，而 ANA 和 FANA 似乎能够模拟与 RNA 形成杂交双链时的 DNA 链几何结构，从而被 RNase H 识别和加工。

2′-Modifications and MOE 2'-修饰与甲氧基乙基

Nucleic acids bearing modifications at the 2′-O-position are among the best studied analogs in the antisense, siRNA and aptamer contexts, starting of course with the 2′-O-methyl modification. One of the reasons the 2′-position has proved so fertile for antisense applications is that modification at that site maintains and potentially enhances preorganization of the sugar for an A-type backbone conformation. Modification also affords chemical stability in that it precludes 2′-OH-mediated strand cleavage. Moreover, it should result in better protection against nuclease attack owing to the vicinity of the 2′-position to the adjacent phosphate.

在反义核酸、siRNA 和适配体研究中，带有 2'-O 位点修饰的核酸是最为广泛研究的类似物之一，当然，最早研究的是 2'-O-甲基修饰。2'-位点在反义应用中被证明非常适合进行修饰的原因之一是该位置的修饰可以维持并潜在增强糖的预组织，使其形成 A 型骨架构象。修饰还可以提供化学稳定性，因为它可以避免 2'-OH 介导的链断裂。此外，由于 2'-位点与相邻磷酸的接近性，它应该能更好地保护核酸免受核酸酶的攻击。

Conformational preorganization produces entropy-based gains in pairing stability and recent work has shed light on the origins of the ca. 1°C increase in Tm per modification seen with 2′-O-Me modified strands opposite RNA. Accordingly, orientations of the 2′-OH group that have the hydrogen directed toward the phosphate group are at the root of the rich tertiary structure of RNA in that this induces non-canonical backbone conformations. Conversely, the 2′-OH hydrogen directed toward the base prevents it from scanning the backbone, thereby stabilizing a canonical (A-form) strand conformation. 2′-O-Me modification mimics the latter situation as it replaces the hydrogen with a methyl group that points away from the backbone and towards the minor groove.

构象预组织产生了配对稳定性的熵增益，最近的研究揭示了 2'-O-甲基修饰链与 RNA 相对时每个修饰引起的T值约 1°C 增加的起源。因此，2'-OH 基团朝向磷酸基团的定向是 RNA 丰富三级结构的根源，因为这会诱导非经典的骨架构象。相反，2'-OH 基团朝向碱基可以阻止其扫描骨架，从而稳定规范（A 型）链的构象。2'-O-甲基修饰模拟了后一种情况，因为它用甲基取代了氢原子，该甲基指向远离骨架并指向较小的凹槽。

We examined the pairing stability and nuclease resistance of ten 2′-O-modifications, including smaller substituents such as propyl, butyl, allyl, 2-fluoroethyl (FET) and 2-(methoxy)ethyl (MOE), as well as bulky, e.g. 2-(benzyloxy)ethyl (BOE), and positively charged moieties, e.g. 2-(imidazol-1-yl)ethyl (IME) and 2-[(N,N-dimethylamino)oxy]ethyl (DMAOE). The nucleobase in most cases was thymine and the tested DNA ASOs contained between three and ten modified nucleotides. The FET, IME and DMAOE modifications stabilized the duplex with RNA most, up to ca. 1.5°C ΔTm per modified nucleotide. By comparison, the MOE modification (Figure 7B) resulted in a ca. 1°C/residue average gain in Tm for the three sequences tested. Interestingly, the ASO with four and ten consecutive BOE modifications still afforded Tm increases of 0.8°C/residue and 0.7°C/residue, respectively.

我们研究了十种 2'-O-修饰的碱基对稳定性和核酸酶的抵抗性，其中包括较小的取代基，如丙基、丁基、烯丙基、2-氟乙基（FET）和 2-(甲氧基)乙基（MOE），以及庞大的取代基，例如 2-(苄氧基)乙基（BOE），和带正电荷的取代基，例如 2-(咪唑-1-基)乙基（IME）和 2-[(N,N-二甲基氨氧基)]乙基（DMAOE）。大多数情况下，碱基是胸腺嘧啶，测试的 DNA ASOs 包含 3 到 10 个修饰的核苷酸。FET，IME 和 DMAOE 修饰使与 RNA 形成的双链稳定性最大，每个修饰核苷酸可使T值增加约 1.5°C。相比之下，MOE 修饰导致三个序列的平均T值增益约为 1°C/核苷酸。有趣的是，具有连续四个和十个 BOE 修饰的 ASO 分别仍然导致 0.8°C/核苷酸和 0.7°C/核苷酸的T值增加。

However, perhaps somewhat unexpectedly given its bulky nature, BOE only provided very little protection against degradation by the 3′-exonuclease snake venom phosphodiesterase (SVPD). In fact, among the investigated modifications, only the 2′-O-propargyl modification was worse. The crystal structure of a 2′-O-BOE-modified duplex revealed that the phenyl rings were positioned relatively far away from the phosphate group. IME, DMAOE and MOE incorporated at the 3′-end were ranked 1–3 in terms of protection against SVPD attack. The higher resistance to nuclease degradation seen with the IME and DMAOE modifications is consistent with the zwitterionic nature of these analogs that may position a positive charge near the 3′-phosphate group. This is consistent with the observation in crystal structures that virtually all 2′-O-substituents were directed toward the 3′-termini of strands.

然而，或许有些出乎意料的是，尽管 BOE 取代基体积庞大，但在面对 3'-外切核酸酶蛇毒磷酸二酯酶（SVPD）的降解时却提供了非常有限的保护。事实上，在所研究的修饰中，只有 2'-O-丙炔基修饰效果更差。2'-O-BOE 修饰的双链结构的晶体结构显示苯环离磷酸基相对较远。以 IME、DMAOE 和 MOE 修饰的 3'-末端为基础，对抗 SVPD 攻击的保护能力分别排名第 1 至第 3 位。IME 和 DMAOE 修饰显示出更高的核酸酶降解抗性，这与这些类似物的带电性质相一致，可能使正电荷靠近 3'-磷酸基。这与晶体结构中的观察结果一致，几乎所有 2'-O-取代基都指向链的 3'-末端。

The MOE-modified nucleoside features extra conformational preorganization as a gauche effect not only dictates the C3′-endo sugar ring pucker but also governs the conformation of the ethylene glycol moiety in the 2′-substituent (Figure 9). A crystal structure of a DNA A-form duplex with a single 2′-O-MOE residue per strand confirmed that the substituents assume a synclinal (sc) conformation. In the crystal structure both MOE-modified nucleosides cradle a water molecule that is coordinated to O3′, O2′ and the outer oxygen atom of the substituent (Figure 9 ( javascript:; )). The structure of a duplex that contained modified residues with a 2′-O-methyl[(tri(oxyethyl)]-substituent (TOE) demonstrated that TOE moieties snake along the backbone, are well ordered and that torsion angles around C-C bonds in the substituent are all in the sc range. In addition to the water molecule associated with MOE substituents, TOE substituents stabilize a second water that coordinates to the two outermost oxygen atoms. Without the gauche effect, conformational preorganization is diminished as seen in the crystal structure of a duplex with incorporated 2′-O-ethoxymethylene (EOM)-modified residues. Unlike MOE- or TOE-modified residues that enhance the thermodynamic stability of a duplex, the EOM modification is destabilizing and the crystal structure showed local unstacking caused by EOM moieties.

MOE 修饰核苷酸具有额外的构象预组织，这是由于"不对称"效应不仅决定了 C3′-内扭糖环的弯曲，还控制了 2′-取代基中乙二醇部分的构象（图 9）。一对一链的 DNA A-型双链结构与单个 2′-O-MOE 残基的晶体结构证实了取代基呈现协轴(sc)构象。在晶体结构中，两个 MOE 修饰核苷酸包裹着一分子水，该水分子与 O3′、O2′和取代基的外部氧原子形成配位键（图 9）。另一个含有 2′-O-甲基[(三(氧乙基)]取代基（TOE）的修饰残基的双链结构显示，TOE 取代基沿着链的骨架排列，有良好的有序性，并且取代基中 C-C 键的扭转角度都在sc范围内。除了与 MOE 取代基相关联的水分子外，TOE 取代基还稳定了第二个与两个最外层氧原子配位的水分子。如果没有"不对称"效应，构象预组织将减弱，这在含有 2′-O-乙氧基亚甲基（EOM）修饰残基的双链结构的晶体结构中可见。与增强双链热力学稳定性的 MOE 或 TOE 修饰残基不同，EOM 修饰是一种降解因素，晶体结构显示由于 EOM 取代基而导致的局部失序现象。

图 9.

A subsequently determined crystal structure of a fully modified MOE–RNA duplex attested further to the importance of conformational preorganization as the underlying cause of the increased stability and nuclease resistance of this analogue . Of the 24 MOE substituents in the duplex 22 adopted the sc+ or sc− conformations and most of them trapped a water in a chelate-like fashion between substituent, sugar and phosphate. Thus, MOE modification can stabilize up to three first-shell water molecules per residue, resulting in extensive hydration networks in the minor groove and around backbones in MOE–RNA.

随后确定的一个完全修饰的 MOE-RNA 双链的晶体结构进一步证实了构象预组织作为增加稳定性和核酸酶抗性的原因的重要性。在该双链中的 24 个 MOE 取代基中，有 22 个采用了sc+或sc−构象，并且其中大多数通过类似螯合的方式将水分子困住在取代基、糖基和磷酸根之间。因此，MOE 修饰可以使每个残基稳定最多三个第一层水分子，从而在 MOE-RNA 的小沟和磷酸骨架周围形成广泛的水合网络。

Oligonucleotides with zwitterionic 2′-O-(3-aminopropyl)-modified (AP, Figure 10) ribonucleotides at the 3′-end displayed extraordinary resistance against degradation by SVPD. To gain insight into the origins of this protective effect, we determined the crystal structure of an AP-modified oligo in complex with E. coli DNA polymerase I 3′-exonuclease Klenow fragment. Rather than just altering the orientation of the substrate strand relative to active site residues, the AP substituent knocked out a catalytic metal ion and engaged in a salt bridge to an aspartic acid side chain. MOE can deliver enhanced pairing stability and nuclease resistance but is no match for the aminopropyl modification in terms of warding off an attack by an exonuclease.

在 3'-末端带有带电性 2'-O-(3-氨基丙基)修饰（AP，图 10）核苷酸的寡核苷酸显示出极高的抗 SVPD 降解能力。为了了解这种保护效应的起源，我们确定了一个 AP 修饰寡聚物与大肠杆菌 DNA 聚合酶 I 3'-外切酶 Klenow 片段的晶体结构。AP 取代基不仅改变了底物链相对于活性位点残基的定位，还排除了一个催化金属离子并与天冬氨酸侧链形成了盐桥。MOE 可以提供增强的配对稳定性和核酸酶抗性，但在抵御外切酶攻击方面无法与氨丙基修饰相媲美。

图 10.

To combine the conformational preorganization of MOE with the record nuclease resistance delivered by the AP substituent, we created the 2′-O-2-[2-(N,N-dimethylamino)ethoxy]ethyl] (DMAEOE) modification (Figure 10). Indeed, DMAEOE affords gains in Tm per modified residue of up to 1.2°C even with stretches of 10 consecutive modified nucleotides in the strand opposite RNA. Consecutive placement of analogues with positively charged 2′-O-substituents, e.g. AP, typically creates Coulombic repulsion in the minor groove with adverse effects on stability. The DMAEOE modification constitutes an exception in this regard as conformational preorganization by the MOE portion prevents entanglement by protonated dimethylamino moieties. Indeed, the crystal structure of a DMAEOE-modified duplex confirmed the hydration motif previously seen with MOE and TOE substituents. Finally, the 2′-O-[2-(guanidinium)ethyl] (GE, Figure 10) modification boosted the stability of ASO-RNA duplexes by 2°C (ΔTm) per modification and the stability of triplexes with duplex DNA by between 2.5 and 4.1°C per modification. This analogue also showed exceptional nuclease resistance but, unlike the findings with DMAEOE, consecutive placement of GE-substituted residues was not tolerated and resulted in a slight loss in Tm of –0.1°C per modified residue.

为了将 MOE 的构象预组织与 AP 取代基提供的出色核酸酶抵抗性相结合，我们设计了 2′-O-2-[2-(N,N-二甲基氨基)乙氧基]乙基 (DMAEOE) 修饰（图 10）。事实上，DMAEOE 即使在与 RNA 相对的链中连续插入 10 个修饰核苷酸时，每个修饰残基的T_m 增益可高达 1.2°C。连续放置具有带正电的 2′-O 取代基的类似物，如 AP，通常在小沟中产生库伦斥力，对稳定性产生不利影响。然而，DMAEOE 修饰在这方面是个例外，因为 MOE 部分的构象预组织防止了质子化二甲基氨基基团的纠缠。实际上，DMAEOE 修饰双链的晶体结构证实了先前在 MOE 和 TOE 取代基中观察到的水合模式。最后，2′-O-[2-(胍基)乙基] (GE，图 10)修饰使 ASO-RNA 双链的稳定性每个修饰增加 2°C (ΔT_m)，并使具有双链 DNA 的三链结构的稳定性每个修饰增加 2.5 到 4.1°C。该类似物也显示出异常的核酸酶抵抗性，但与 DMAEOE 的研究结果不同，连续放置的 GE 取代残基不能被耐受，并导致每个修饰残基的T_m轻微降低-0.1°C。

KYNAMRO, TEGSEDI and WAYLIVRA

Since the original synthesis and characterization in 1995, MOE–RNA has been the focus of numerous investigations regarding its clinical efficacy, culminating in the approval of three MOE–RNA/PS–DNA ASO gapmers, KYNAMRO (mipomersen) , TEGSEDI (inotersen) and WAYLIVRA (volanesorsen) (Figure 11). The plasma pharmacokinetics (PK) following intravenous (i.v.) administration of an all-PS 5–10–5 ASO with MOE wings targeting human tumor necrosis factor α were characterized in mouse, rat, dog, monkey and human and found to exhibit quite rapid distribution with a half-life of between 15 and 45 min in all species. Subcutaneous injection of the ASO resulted in absorptions of 80–100%, and when the oligo was administered intrajejunally, bioavailability reached 10% compared to i.v. administration. In monkeys less than 1% of the ASO administered by i.v. at up to 5 mg/kg was excreted in urine, the high levels of plasma protein-bound oligo presumably preventing renal filtration. In all species elimination of the ASO from tissue was very slow (several days) and dependent on tissue and organ, whereby the highest concentrations were seen in kidney, liver, lymph nodes, spleen and bone marrow. In a further plasma PK study, tissue distribution and metabolism of three ASOs with a phosphodiester (PO) backbone, all-PS and MOE at both 5′- and 3′-end, or all-PS but with MOE only at the 3′-end were compared after i.v. infusion of a 10 mg/kg dose in monkeys. Plasma clearance was significantly higher for the PO oligos compared to the two PS ones and was attributed to reduced plasma protein binding and nuclease resistance.

自从 1995 年首次合成和表征以来，MOE-RNA 一直是许多临床研究的焦点，最终批准了三种 MOE-RNA/PS-DNA ASO gapmer，分别是 KYNAMRO（mipomersen）、TEGSEDI（inotersen）和 WAYLIVRA（volanesorsen）（图 11）。通过静脉注射给予针对人类肿瘤坏死因子α的全 PS 5-10-5 ASO 与 MOE 翅膀，对小鼠、大鼠、狗、猴和人体进行了血浆药代动力学（PK）研究，发现在所有物种中都表现出相当快速的分布，半衰期在 15 到 45 分钟之间。皮下注射 ASO 导致 80-100%的吸收，而当寡核苷酸经肠内注射时，生物利用度相对于静脉注射达到 10%。在猴子中，通过静脉注射给予高达 5 mg/kg 的 ASO，不到 1%的剂量在尿液中排出，高水平的血浆蛋白结合寡核苷酸可能阻止了肾脏过滤。在所有物种中，ASO 从组织中的消除速度非常缓慢（几天），并且取决于组织和器官，其中最高浓度出现在肾脏、肝脏、淋巴结、脾脏和骨髓中。在进一步的血浆 PK 研究中，比较了具有磷酸二酯（PO）骨架、全 PS 和 MOE 在 5'-和 3'-端的 ASO 以及全 PS 但只在 3'-端具有 MOE 的三种 ASO 在猴子中 10 mg/kg 剂量的静脉输注后的组织分布和代谢情况。与两种 PS ASO 相比，PO 寡核苷酸的血浆清除率显著更高，这归因于减少的血浆蛋白结合和核酸酶抵抗性。

图 11.

The ASO with MOE modification at both ends exhibited greatly enhanced nuclease resistance in plasma and tissues, whereas the one with MOE modification only at the 3′-end resisted degradation in plasma to a certain degree but displayed only moderate resistance in tissues. Urinary excretion was a major elimination pathway for the PO oligo, but accounted for only a minor role in the elimination of the two PS/MOE-modified ASOs. The in vitro metabolic stabilities of PO and all-PS ASOs with MOE wings were also studied in a whole liver homogenate from rat or human and the outcomes including the metabolites compared to in vivo half-lives. The metabolites identified in vitro were consistent with those from in vivo observations and the relative stabilities of PO and PS/MOE oligos were similar in vitro and in vivo. However, the in vitro assay had limited predictive value in terms of the differences in metabolic stability exhibited by individual MOE/PS oligos. The metabolic pathway inferred from analysis of the metabolites in the preincubated liver homogenates was consistent with an initial endonucleolytic attack in the middle of the central PS–DNA window, followed by 5′- and 3′-exonuclease action. Among all investigated analogues, MOE–RNA constitutes the most thoroughly investigated modification with extensive clinical trial data available.

在血浆和组织中，两端均含有 MOE 修饰的 ASO 表现出极强的核酸酶抗性，而仅在 3'-端含有 MOE 修饰的 ASO 在血浆中抵抗降解到一定程度，但在组织中只表现出中等的抗性。尿排泄是 PO 寡核苷酸的主要排除途径，但对两种 PS/MOE 修饰的 ASO 的排除作用只起到次要的作用。还研究了含有 MOE 翅膀的 PO 和全 PS ASO 在大鼠或人类整个肝脏均匀液中的体外代谢稳定性，将其结果与体内半衰期及代谢物进行比较。体外鉴定的代谢物与体内观察的代谢物一致，PO 和 PS/MOE 寡核苷酸的相对稳定性在体内外相似。然而，体外测定对于 MOE/PS 寡核苷酸的代谢稳定性差异具有有限的预测价值。通过对预孵肝脏均匀液中的代谢物进行分析，推断出代谢途径，首先在中央 PS-DNA 窗口的中部进行内切核酸酶攻击，然后进行 5'-和 3'-外切核酸酶作用。在所有研究的类似物中，MOE-RNA 是最经彻底研究的修饰，拥有广泛的临床试验数据。

KYNAMRO targeted the mRNA of ApoB protein, reduced cholesterol and was approved for treatment of familial hypercholesterolemia. It was the first systemically delivered ASO that obtained market approval (2013). KYNAMRO was given by injection once a week and lowered LDL-C levels in patients, but adverse effects included hepatotoxicity and injection site reactions. JUXTAPID® (lomitapide), a small molecule drug that, like KYNAMRO, comes with a risk of hepatotoxicity, ultimately proved more successful commercially, perhaps, in part, due to its oral administration.

KYNAMRO 以靶向 ApoB 蛋白的 mRNA 为目标，降低胆固醇，并获得了治疗家族性高胆固醇血症的批准。它是第一个获得市场批准的系统给药 ASO（2013 年）。KYNAMRO 每周注射一次，可降低患者的低密度脂蛋白胆固醇（LDL-C）水平，但不良反应包括肝毒性和注射部位反应。JUXTAPID®（洛美他派），一种小分子药物，与 KYNAMRO 一样，存在肝毒性风险，但在商业上取得了更大的成功，可能部分原因是其口服给药形式。

TEGSEDI is an ASO that targets the transthyretin (TTR) 3′-UTR and thereby inhibits production of wild-type and mutant TTR proteins. TTR acts as the primary transporter of retinol-binding protein and is also a redundant transport protein for thyroxine. The protein forms a homo-tetramer but mutations in the TTR monomer – there are some 150 reported mutations – destabilize the tetrameric structure. Dissociation of the tetramer releases misfolded monomers that can associate to form toxic oligomers and amyloid fibrils. Deposition of these TTR amyloid fibrils is referred to as TTR amyloidosis (ATTR) and the hereditary form of the autosomal dominant disorder is called hATTR. The predominant manifestations of the disease are either polyneuropathy or cardiomyopathy.

TEGSEDI 是一种靶向甲状腺素转运蛋白（TTR）3'-非翻译区（3'-UTR）的 ASO，从而抑制野生型和突变型 TTR 蛋白的产生。TTR 是维生素 A 结合蛋白的主要运输蛋白，也是甲状腺素的冗余运输蛋白。该蛋白形成一个同源四聚体，但 TTR 单体中的突变（已报道约 150 种突变）会使四聚体结构不稳定。四聚体的解离释放出错误折叠的单体，可以聚集形成有毒寡聚体和淀粉样纤维。这些 TTR 淀粉样纤维的沉积被称为 TTR 淀粉样病变（ATTR），而遗传性的常染色体显性遗传疾病形式被称为 hATTR。该疾病的主要表现为多发性神经病或心肌病。

Therapeutic intervention for hATTR in the US prior to 2018 included liver transplantation, stabilizing the TTR tetramer (diflunisal), or reducing TTR deposition with a combination of doxycycline and tauroursodeoxycholic acid. The first siRNA for hATTR treatment, ONPATTRO was approved in 2018 for the treatment of the polyneuropathy of hATTR mediated amyloidosis in adults. The antisense oligonucleotide TEGSEDI was approved later in the same year for the same indication. The two drugs have different routes of administration, and different safety profiles. TEGSEDI is administered subcutaneously, whereas ONPATTRO is administered intravenously as an LNP formulation.

2018 年之前，美国对 hATTR 的治疗干预方法包括肝移植、稳定 TTR 四聚体（地氟尼索）或使用多西环素和牛磺胆酸组合减少 TTR 沉积。2018 年，首个用于治疗 hATTR 的小干扰 RNA（siRNA）药物 ONPATTRO 获得批准，用于治疗成年人 hATTR 介导的多发性神经病。同年稍后，用于同一适应症的反义寡核苷酸药物 TEGSEDI 也获得批准。这两种药物具有不同的给药途径和不同的安全性特点。TEGSEDI 通过皮下给药，而 ONPATTRO 通过静脉注射作为 LNP 制剂给药。

WAYLIVRA is an ASO that targets the 3′-untranslated region (3′-UTR) of apolipoprotein C III (apoC III) mRNA and was approved by the European Union to treat familial chylomicronemia syndrome (FCS) in 2019. FCS is a metabolic disorder, causes hypertriglyceridaemia that involves a functionally impaired lipoprotein lipase (LPL) and is associated with potentially fatal consequences such as pancreatitis. The disorder is characterized by chylomicronemia and hypertriglyceridemia resulting from LPL action. ApoC III inhibits LDL and increases the concentration of plasma triglyceride by lowering the hepatic uptake of triglyceride-rich lipoproteins while also raising hepatic secretion of triglycerides. The drug is administered by subcutaneous injection; adverse effects include mild injection site reactions, a decreased platelet count and thrombocytopenia. In further clinical trials the utility of WAYLIVRA was assessed for the treatments of hypertriglyceridemia, familial partial lipodystrophy and partial lipodystrophy. However, it doesn’t appear in the current pipeline and was replaced by the corresponding GalNAc conjugate.

WAYLIVRA 是一种靶向载脂蛋白 C III（apoC III）mRNA 的 3'-非翻译区（3'-UTR）的反义寡核苷酸药物，于 2019 年获得欧盟批准，用于治疗家族性乳糜微粒血症综合征（FCS）。FCS 是一种代谢性疾病，导致高甘油三酯血症，其中涉及功能受损的脂蛋白脂肪酶（LPL），并且与胰腺炎等潜在致命后果相关。该疾病的特征是由于 LPL 作用引起的乳糜微粒血症和高甘油三酯血症。apoC III 通过降低肝脏对富含甘油三酯的脂蛋白的摄取而增加血浆甘油三酯浓度，同时抑制低密度脂蛋白（LDL）的作用并提高肝脏甘油三酯的分泌。该药物通过皮下注射给药；不良反应包括轻度注射部位反应、血小板计数降低和血小板减少症。在进一步的临床试验中，评估了 WAYLIVRA 在治疗高甘油三酯血症、家族性部分脂肪营养不良和部分脂肪营养不良方面的功效。然而，该药物目前不在当前的研发计划中，并被相应的 GalNAc 结合物所取代。