Martin Egli, Muthiah Manoharan
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Lipid nanoparticles 脂质纳米粒
The first two siRNA therapeutics to receive market approval, ONPATTRO and GIVLAARI, use entirely different delivery systems, LNP carrier versus triantennary carbohydrate conjugate to target ASGPR, respectively (Figure 20). Both approaches serve to deliver the siRNA duplex to the cytoplasm of hepatocytes in vivo. The development of efficient delivery systems is key to the success of oligonucleotide therapeutics and remains a very active area of research. Several excellent reviews of the oligonucleotide delivery field have been published in recent years and the interested reader may turn to these for more in-depth discussions of the various challenges and strategies being pursued to address them including oral delivery.
获得市场批准的第一批 siRNA 药物 ONPATTRO 和 GIVLAARI 使用了完全不同的递送系统，分别为 LNP 载体和三支糖链偶联物以靶向 ASGPR（图 20）。这两种方法都用于将 siRNA 双链递送到体内肝细胞的细胞质中。高效的递送系统的开发对寡核苷酸治疗药物的成功至关重要，并且仍然是一个非常活跃的研究领域。近年来已经发表了一些对寡核苷酸递送领域进行了出色综述的文章，感兴趣的读者可以参考这些综述以获取更深入的讨论，了解应对挑战的各种策略，包括口服递送。
Briefly, a first hurdle to overcome is getting the oligo to the particular tissue of therapeutic interest. The obstacles tissues have erected that need to be scaled include the vascular endothelial barrier, the reticuloendothelial system, renal excretion and its effects both on pharmacokinetics and biodistribution, as well as the blood-brain barrier. Receptor-mediated targeting may allow cell-specific delivery and internalization of the oligos. Examples of receptor families are integrins, G protein-coupled receptors, receptor tyrosine kinases, Toll-like receptors, scavenger receptor, folate receptor, and, as mentioned before, ASGPR. But after reaching cell surface and internalization by endocytosis, an oligonucleotide remains separated from the cytosol and thus its ultimate target if it cannot escape the endosome. Therefore, understanding the intracellular trafficking machinery and breaching the endosomal barrier critically affect the design and clinical success of oligonucleotide therapeutics. There are many other delivery approaches besides the aforementioned LNPs and GalNAc conjugates for targeting ASGPR, but because they proved successful first in the clinic, we will limit the discussion to these two in this review and perspective article.
克服的第一个障碍是将寡核苷酸送达到特定的治疗目标组织。需要克服的障碍包括血管内皮屏障、网状内皮系统、肾脏排泄以及对药代动力学和生物分布的影响，以及血脑屏障。受体介导的靶向可能实现寡核苷酸的细胞特异性递送和内吞。一些受体家族的例子包括整合素、G 蛋白偶联受体、受体酪氨酸激酶、Toll 样受体、清道夫受体、叶酸受体，以及前面提到的 ASGPR。但是，在达到细胞表面并通过内吞作用内化之后，如果寡核苷酸不能逃脱内体，它将与细胞质和最终目标分离。因此，了解细胞内的运输机制和突破内体屏障对寡核苷酸治疗药物的设计和临床成功至关重要。除了前面提到的脂质体纳米粒和 GalNAc 偶联物以靶向 ASGPR 之外，还有许多其他递送方法。但是，由于它们在临床中取得了成功，本文将限制讨论这两种递送方法。
LNPs represent important delivery systems for siRNA therapeutics and the particular lipids and lipoids used in the multi-component formulation of the carrier for ONPATTRO are shown in Figure 21A. The second generation ionizable amino-lipid heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA) afforded increased potency compared to first generation benchmark LNPs. In the positively charged state this lipid interacts with negatively charged lipids, thereby disrupting the bilayer membrane and allowing release of the siRNA from the endosome into the cytoplasm. Another key component is the PEG lipid mPEG2000-C14 glyceride that is associated with the surface of the LNP and influences the diameter of the particle in a concentration-dependent manner. PEG lipids can dissociate from the LNP and bind to lipoprotein particles in plasma which facilitates interactions between the unshielded LNP and target cells and thus enables uptake. Two additional components of the LNP are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (Figure 21A). It was recently shown that DSPC-cholesterol resides in the outer layers when the LNP is empty, i.e. not containing the siRNA. However, once siRNAs are loaded into the system, DSPC-cholesterol becomes internalized together with siRNA, thus suggesting that they act as helper lipids and are key to stable encapsulation of the RNA cargo.
脂质体纳米粒（LNPs）是 siRNA 药物的重要递送系统，ONPATTRO 的载体的多组分配方中使用的特定脂质和脂质类物质如图 21A 所示。第二代离子化氨基脂质 heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate（DLin-MC3-DMA）与第一代基准 LNP 相比具有更高的效力。在带正电的状态下，该脂质与带负电的脂质发生相互作用，从而破坏双层膜，并允许 siRNA 从内体释放到细胞质中。另一个关键组分是 PEG 脂质 mPEG2000-C14 甘油酯，它与 LNP 的表面相关，并以浓度依赖的方式影响粒子的直径。PEG 脂质可以从 LNP 中解离，并结合到血浆中的脂蛋白粒子上，从而促进未被屏蔽的 LNP 与靶细胞之间的相互作用，从而实现摄取。LNP 的另外两个组分是 1,2-二硬脂酸-sn-甘油-3-磷酰胆碱（DSPC）和胆固醇（图 21A）。最近的研究表明，在 LNP 为空的情况下，DSPC-胆固醇存在于外层。然而，一旦 siRNA 被加载到系统中，DSPC-胆固醇会与 siRNA 一起内化，这表明它们起到辅助脂质的作用，并且对于稳定封装 RNA 荷载至关重要。
The mechanistic model of LNP-mediated delivery of ONPATTRO into hepatocytes entails the following steps. (i) PEG lipids are released from the particle surface. (ii) Endogenous apolipoprotein E (ApoE) is recruited to the LNP surface. (iii) ApoE promotes trafficking of LNPs through fenestrated endothelium and subsequent binding to lipoprotein receptors on the surface of hepatocytes. (iv) Internalization of LNPs via endocytosis. (v) The low pH in the endosome results in protonation of DLin-MC3-DMA. (vi) Interactions between positively charged DLin-MC3-DMA and negatively charged endogenous lipids destabilize the endosomal membrane. (vii) siRNA is released into the cytoplasm of hepatocytes and loaded into Ago2-RISC.
ONPATTRO 通过 LNP 介导的递送机制进入肝细胞，包括以下步骤：（i）PEG 脂质从粒子表面释放出来。（ii）内源性载脂蛋白 E（ApoE）被招募到 LNP 表面。（iii）ApoE 促进 LNPs 通过有孔内皮细胞的运输，并随后结合到肝细胞表面的脂蛋白受体上。（iv）通过内吞作用将 LNPs 内化。（v）内体液泡中的低 pH 使 DLin-MC3-DMA 质子化。（vi）正电荷的 DLin-MC3-DMA 与负电荷的内源性脂质之间的相互作用破坏内体液泡膜。（vii）siRNA 释放到肝细胞的细胞质中，并加载到 Ago2-RISC 中。
Asialoglycoprotein receptor and GalNAc conjugates 去唾液酸糖蛋白受体和 GalNAc 偶联物
Chemical modification of oligonucleotides affects their physical-chemical properties and we discussed the effects of PS modification on pairing stability, nuclease resistance, protein binding, cellular uptake and pharmacokinetics and pharmacodynamics. Moreover, uptake and biodistribution of single-stranded ASOs and double-stranded siRNAs used alone versus in the presence of a carrier, e.g. LNPs, are obviously quite different and siRNAs face bigger hurdles with respect to delivery. Conjugation of a chemically modified oligonucleotide to a ligand potentially offers a strategy to overcome the challenges facing delivery of a polyanion of molecular weight >10 kDa across a cell membrane.
寡核苷酸的化学修饰会影响其物理化学性质，我们已经讨论了磷酸酯（PS）修饰对配对稳定性、核酸酶抗性、蛋白结合、细胞摄取以及药代动力学和药效学的影响。此外，单链反义寡核苷酸（ASO）和双链小干扰 RNA（siRNA）在单独使用和与载体（例如 LNPs）共同使用时的摄取和生物分布显然是不同的，siRNA 在传递方面面临着更大的障碍。将化学修饰的寡核苷酸与配体结合可能是克服将分子量大于 10 kDa 的多阴离子通过细胞膜传递的挑战的一种策略。
In pioneering work dating back >30 years, Letsinger and colleagues reported the synthesis, properties and activity of cholesteryl-conjugated oligos targeted against several HIV proteins in cell culture. The cholesterol moiety was tethered to the 3′-end of the oligos and significantly increased their antiviral potencies. The hydrophobic cholesterol conjugation was also shown to improve interactions between siRNA and plasma lipoproteins, uptake and tissue distribution. Cholesterol promotes enhanced association of oligos with low-density or high-density lipoproteins (LDL or HDL, respectively).
在 30 多年前的开创性研究中，Letsinger 及其同事报告了合成、性质和活性与几种 HIV 蛋白靶向的胆固醇偶联寡核苷酸在细胞培养中的研究。胆固醇部分被连接到寡核苷酸的 3'-末端，显著增强了其抗病毒效能。疏水性的胆固醇偶联还改善了 siRNA 与血浆脂蛋白的相互作用、摄取和组织分布。胆固醇促进了寡核苷酸与低密度脂蛋白（LDL）或高密度脂蛋白（HDL）的增强结合。
Although hepatic targeting is most commonly pursued with cholesterol-conjugated siRNAs, delivery of siRNA to muscle and other extrahepatic tissues has also been demonstrated with cholesterol conjugation chemistry. A comprehensive study compared fifteen different lipid-conjugation chemistries with regards to siRNA tissue distribution and efficacy. In general, the hydrophobicity of the conjugate correlated with the degree of clearance and the distribution between liver and kidney. One notable finding was that several of the conjugates afforded accumulation of siRNA in extra-hepatic tissues such as muscle, lung, heart and adrenal glands. Moreover, cholesterol was inferior to several of the tested conjugates in promoting siRNA distribution to extra-hepatic tissues. This suggests that the chemical nature of the lipid conjugate plays an important role in enabling and maximizing siRNA delivery to organs other than liver. Current efforts directed at the development and clinical use of siRNA-bioconjugates have recently been reviewed. The bioconjugation platform for siRNA delivery is rapidly expanding and besides the abovementioned lipids, conjugates tethered to RNA termini include peptides, receptor ligands, aptamers, antibodies, and CpG oligos.
虽然胆固醇偶联 siRNA 通常用于肝脏靶向，但也已经通过胆固醇偶联化学方法将 siRNA 传递到肌肉和其他肝外组织中。一项综合研究比较了 15 种不同的脂质偶联化学方法在 siRNA 组织分布和效果方面的差异。总体而言，偶联物的疏水性与清除程度和肝脏与肾脏之间的分布相关。一个值得注意的发现是，其中几种偶联物导致 siRNA 在肌肉、肺、心脏和肾上腺等肝外组织中的积累。此外，胆固醇在促进 siRNA 分布到肝外组织方面不如其他被测试的偶联物。这表明脂质偶联物的化学性质在实现和最大化 siRNA 传递到肝脏以外器官中起着重要作用。最近对 siRNA 生物偶联物的开发和临床应用进行了综述。siRNA 传递的生物偶联平台正在迅速扩展，除了上述脂质偶联物外，还包括与 RNA 末端相连的肽、受体配体、适配体、抗体和 CpG 寡核苷酸。
The triantennary GalNAc conjugate tethered to the 3′-terminus of the GIVLAARI siRNA passenger strand enables efficient ASGPR-mediated delivery of the therapeutic to hepatocytes. ASGPR is abundantly expressed in hepatocytes and congregates on the cell surface in coated pits. The receptor can form a trefoil composed of ASGPR1 and ASGPR2 subunits (called H1 and H2, respectively, in the human system), whereby the former features a carbohydrate recognition domain (CRD; Figure 22). Crystal structures of H1-CRD alone and bound to either lactose or a bicyclic bridged ketal that efficiently binds to ASGPR were determined. In addition, the crystal structure of a mannose binding protein mutant in complex with GalNAc provided insight into the preferential binding of ASGPR-CRD to this sugar compared with galactose (Figure 22).
三叉状的 GalNAc 偶联物与 GIVLAARI siRNA 载体链的 3'-末端相连，实现了通过 ASGPR 介导的有效治疗物传递到肝细胞。ASGPR 在肝细胞中丰富表达，并在细胞表面聚集于包被窝。该受体可以形成由 ASGPR1 和 ASGPR2 亚单位组成的三叉形结构（在人体系统中分别称为 H1 和 H2），其中前者具有碳水化合物识别结构域（CRD；图 22）。已确定了 H1-CRD 单独和与乳糖或有效结合 ASGPR 的双环桥酮结合时的晶体结构。此外，一个与甘露糖结合蛋白突变体与 GalNAc 结合形成的晶体结构揭示了相比于半乳糖，ASGPR-CRD 与甘露糖的优先结合性（图 22）。
图 22.
The carbohydrate binding site is shallow and exposed to solvent and this may explain why multiple subunits are needed to confer sufficient binding affinity and selectivity for the GalNAc sugar. Manoharan and coworkers designed modified siRNAs with trivalent GalNAc conjugates tethered to the 3′-end of the passenger strand. Careful optimization of the chemistry connecting the terminus of the passenger to the linker, the linker chemistry, and the spacers in the triantennary portion of the conjugate resulted in the current ‘L96’ design (Figure 22). Thus, the 3′-terminal phosphate is attached to a prolinol sugar and a C12-spacer connects via amide moieties prolinol sugar on one side and trivalent spacer and GalNAc sugars on the other. In the L96 conjugate, the spacers also contain two amide groups that interrupt the carbon chain and provide some polarity and conformational rigidity.
糖结合位点是浅层且暴露于溶剂中，这可能解释了为什么需要多个亚单位才能赋予足够的结合亲和力和选择性来与 GalNAc 糖发生作用。Manoharan 和他的同事设计了将三糖基 GalNAc 偶联物连接到乘客链的 3'-端的修饰 siRNA。通过仔细优化连接乘客链末端的化学结构、连接物的化学结构以及三糖基部分的间隔物，得到了目前的"L96"设计（图 22）。因此，3'-末端的磷酸根与脯氨醇糖和 C12 间隔物通过酰胺基团相连，其中一侧是脯氨醇糖，另一侧是三糖基间隔物和 GalNAc 糖。在 L96 偶联物中，间隔物还含有两个酰胺基团，中断了碳链并提供了一定的极性和构象刚性。
The GalNAc conjugation chemistry for ASGPR-mediated hepatic delivery and successfully applied in the case of GIVLAARI to treat acute hepatic porphyria is now also being tested with other siRNAs and ASOs currently in the clinic. A recent review listed no fewer than 9 siRNAs and 12 ASOs with GalNAc conjugates in preclinical, phase 1 or phase 2 trials.
ASGPR 介导的肝脏传递所采用的 GalNAc 偶联化学方法已成功应用于 GIVLAARI 治疗急性肝性卟啉症，并正在测试其他目前正在临床试验中的 siRNA 和 ASO。最近的一篇综述列出了不少于 9 个 siRNA 和 12 个 ASO，它们采用了 GalNAc 偶联物在临床前、一期或二期试验中进行研究。
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Nucleic acid aptamers are single-stranded, folded RNA or DNA oligonucleotides that bind molecular targets (small molecules, proteins or nucleic acids) with high specificity and affinity. Aptamers against diverse targets can be identified from oligonucleotide libraries of sufficient sequence complexity by a process called ‘systematic evolution of ligands by exponential enrichment’ . They hold great potential in therapeutics as highly specific modulators of gene function as well as in diagnostics. As in the case of ASOs and siRNAs, native RNA and DNA aptamers are unsuitable for applications in biological systems because of rapid degradation in vitro and in vivo. They also have poor pharmacokinetic properties as putative therapeutics in vivo. Importantly, searching only sequence space often yields aptamers that are limited in specificity and particularly in affinity. Beyond sequence space, it is therefore necessary to explore the chemical and structure space to identify more mature functional aptamers.
核酸适配体是单链折叠的 RNA 或 DNA 寡核苷酸，具有高度特异性和亲和力，可与小分子、蛋白质或核酸等分子靶点结合。通过一种称为“指数富集下的适配体系统进化”的过程，可以从具有足够序列复杂性的寡核苷酸文库中鉴定出针对不同靶点的适配体。它们在基因功能的高度特异性调节和诊断方面具有巨大潜力。与 ASO 和 siRNA 的情况类似，由于在体外和体内快速降解，原生 RNA 和 DNA 适配体不适用于生物系统中的应用。它们在体内作为潜在的治疗药物也具有较差的药代动力学特性。重要的是，仅在序列空间中搜索通常会得到具有限特异性和亲和力的适配体。因此，除了序列空间外，还有必要探索化学和结构空间以寻找更成熟的功能性适配体。
MACUGEN (pegaptanib) was the first RNA-based therapeutic approved by the US FDA (2004, Figures 2and 3) and is the only aptamer among oligonucleotide drugs that have reached the market to date. The 27-nucleotide aptamer targets VEGF for the treatment of wet age-related macular degeneration. It is composed entirely of 2′-F and 2′-O-Me modified nucleotides except for two residues (Figure 23A). Vascular endothelial growth factor (VEGF) is a prominent drug target and extensive studies confirmed a role for VEGF in ocular neovascular diseases and demonstrated that VEGF165 is the isoform largely responsible for pathological ocular neovascularization. Although both VEGF165 and VEGF121 are effective mitogens for human umbilical vein endothelial cells, pretreatment of cells with active ingredient inhibited proliferative responses only to VEGF165. These findings are consistent with photo-crosslinking experiments that demonstrated that binding of pegaptanib to VEGF involves a close contact with Cys-137 of VEGF165. This residue is contained within the 55-amino-acid heparin-binding domain (HBD) of VEGF (Figure 23B) which is not present in VEGF121.
MACUGEN（pegaptanib）是美国 FDA 批准的第一种 RNA 类药物（2004 年，图 2 和 3），也是迄今为止进入市场的寡核苷酸药物中唯一的适配体。这种由 27 个核苷酸组成的适配体针对 VEGF，用于治疗湿性年龄相关性黄斑变性。除了两个残基外，它完全由 2'-F 和 2'-O-Me 修饰的核苷酸组成（图 23A）。血管内皮生长因子（VEGF）是一个重要的药物靶点，广泛的研究证实了 VEGF 在眼部新生血管性疾病中的作用，并表明 VEGF165 是主要导致病理性眼部新生血管化的亚型。虽然 VEGF165 和 VEGF121 对人脐静脉内皮细胞具有促增殖作用，但用活性成分对细胞进行预处理仅抑制对 VEGF165 的增殖反应。这些发现与光交联实验一致，该实验表明 pegaptanib 与 VEGF 的结合涉及与 VEGF165 的 Cys-137 的密切接触。这个残基位于 VEGF 的 55 个氨基酸的肝素结合区域（HBD）内（图 23B），而 VEGF121 中则没有这个结构域。
图 23.
Exploration of sequence space using SELEX resulted in the initial anti-VEGF RNA aptamer. Subsequent trial-and-error exploration of the ‘chemical space’ to optimize the RNA sequence resulted in the high-affinity pegaptanib aptamer with a Kd of 49 pM. One limitation of the incorporation of 2′-F modified nucleotides was that only the pyrimidines were commercially available at the time. Hence the use of the 2′-O-Me modification chemistry for purine residues (Figure 23A). Another important modification concerns PEGylation at the 5′-end that confers drug-like properties to the aptamer for uptake and biodistribution; MACUGEN was administered by intravitreal injection. In principle, a certain amount of chemical modification can be built into the selection process, as in the case of a procedure to identify all-2′-O-Me RNA aptamers against VEGF (Figure 23C). Such aptamers bound VEGF with equilibrium dissociation constants of 1–4 nM and were reasonably stable to degradation in serum. However, these affinities are significantly worse than the pM value for MACUGEN. Because pM binding affinities are highly desirable for increased potency in a therapeutic use this difference is noteworthy. For comparison, LUCENTIS® (ranibizumab), a monoclonal antibody-based therapeutic for wet AMD has a VEGF binding affinity of 140 pM. Antibody-based approaches have become the most prescribed therapies for AMD treatment and have all but replaced MACUGEN.
通过 SELEX 对序列空间的探索得到了最初的抗 VEGF RNA 适配体。随后通过“化学空间”的试错探索优化了 RNA 序列，得到了具有 49 pM 的高亲和力的 pegaptanib 适配体。2'-F 修饰核苷酸的一个限制是当时仅有嘧啶类可供商业使用。因此，使用 2'-O-Me 修饰化学对嘌呤残基进行修饰（图 23A）。另一个重要的修饰涉及 PEG 化在 5'-末端，使适配体具有类似药物的特性，用于摄取和生物分布；MACUGEN 通过眼内注射给予患者。原则上，一定程度的化学修饰可以纳入选择过程中，例如通过一种识别 VEGF 的全 2'-O-Me RNA 适配体的过程（图 23C）。这些适配体与 VEGF 结合的平衡解离常数为 1-4 nM，并在血清中相对稳定。然而，这些亲和力明显不及 MACUGEN 的 pM 级别。因为 pM 级别的结合亲和力在治疗中增加药效方面非常理想，这种差异值得注意。作为对比，作为湿性 AMD 的单克隆抗体治疗药物的 LUCENTIS®（ranibizumab）具有 140 pM 的 VEGF 结合亲和力。基于抗体的方法已成为 AMD 治疗中使用最广泛的疗法，并且几乎已经取代了 MACUGEN。
Many approaches have been utilized to try and improve aptamer affinity including chemical modification. Thus, selection of aptamers against interleukin-6 (IL-6) from a library of DNA molecules containing 40 random positions and 5-(N-benzylcarboxamide)-2′-deoxyuridine (BndU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (NapdU) replacing dT furnished candidates of improved binding (10-fold; relative to DNA) and inhibition (20-fold), but overall modest affinity (Kd = 0.2 nM) and potency (IC50 = 0.2 nM). Further post-SELEX modification placing 2′-O-Me at certain sites improved the affinity minimally. Moreover, this selection process approach to enhance the hydrophobic contributions to the aptamer-protein target interface is laborious, expensive, and limited to modifications tolerated by the polymerases employed.
为了提高适配体的亲和力，人们采用了许多方法，包括化学修饰。例如，通过从包含 40 个随机位点和 5-（N-苄基甲酰胺）-2'-脱氧尿嘧啶（BndU）或 5-[N-（1-萘甲基）甲酰胺]-2'-脱氧尿嘧啶（NapdU）的 DNA 分子库中选择针对白细胞介素-6（IL-6）的适配体，得到了亲和力改善（相对于 DNA，提高了 10 倍）和抑制效果增强（提高了 20 倍）的候选物，但总体亲和力（Kd = 0.2 nM）和效力（IC50 = 0.2 nM）仍然相对较低。进一步在 SELEX 之后的修饰中引入 2'-O-Me 在特定位点上略微提高了亲和力。此外，这种选择过程中用于增强适配体-蛋白质靶标界面的疏水性贡献的方法费时、费钱，并且受到所使用的聚合酶所容忍的修饰的限制。
We demonstrated that a single phosphorodiothiate (PS2) modification can boost the affinity of RNA aptamers against VEGF and thrombin 1000-fold and tighten the binding affinity to 1–2 pM (Figure 23C). Comparison of crystal structures for complexes between thrombin and the native and PS2-modified RNA aptamers and subsequent calculations identified three components that underlie the remarkable change in binding affinity enabled by a single PS2 modification. (i) A hydrophobic patch (phenylalanine, Phe) at the floor of a slight depression on the surface of the protein target. (ii) Local flexing of the RNA backbone that moves the PS2 moiety significantly closer to the edge of the phenyl ring compared to the native phosphate-Phe pair. (iii) An electric field generated by pairs of lysines and arginines that surround the hydrophobic patch and result in polarization of the sulfur that interacts with Phe.
我们证明了单个磷酸二酯硫（PS2）修饰能够将针对 VEGF 和凝血酶的 RNA 适配体的亲和力提高 1000 倍，并将结合亲和力提高到 1-2 pM 的水平（图 23C）。通过比较凝血酶与天然和 PS2 修饰的 RNA 适配体之间的复合物的晶体结构和随后的计算，我们确定了导致单个 PS2 修饰引发的亲和力显著改变的三个组分：（i）位于蛋白质靶标表面轻微凹陷处的疏水性区域（苯丙氨酸，Phe）；（ii）RNA 骨架的局部弯曲使得 PS2 基团与苯环边缘的距离明显缩短，相比天然磷酸-Phe 对；（iii）由赖氨酸和精氨酸成对产生的电场环绕疏水性区域，并导致与 Phe 相互作用的硫原子极化。
One disadvantage of the VEGF system in regard to optimization of aptamer affinity and selectivity is that, unlike for other aptamers including the anti-thrombin RNA, the 3D structures of VEGF165 and those of the native and chemically modified anti-VEGF RNA aptamers are not known. Thus, structural information regarding the components of the complex is limited to the NMR solution structure of the VEGF-HBD. Moreover, it was demonstrated by NMR that the secondary structures of the aptamer are maintained between the complexes with VEGF165 and VEGF-HBD (55 residues) .
在优化 aptamer 的亲和力和选择性方面，与其他 aptamer（包括抗凝血酶 RNA）不同的是，VEGF 系统存在一个不利因素，即 VEGF165 及其天然和化学修饰的抗 VEGF RNA aptamer 的三维结构尚未知晓。因此，关于复合物组分的结构信息仅限于 VEGF-HBD 的 NMR 溶液结构。此外，NMR 实验证明 aptamer 在与 VEGF165 和 VEGF-HBD（55 个残基）形成复合物时，其二级结构得以保持。
A recent exciting development is SELEX with adapted polymerases and xeno nucleic acids (XNAs) . Aptamers based on 2′-F-arabinonucleic acid (FANA), (3′→2′)-L-α-threofuranosyl nucleic acid (TNA) and other XNAs are highly resistant to degradation and offer the prospect of expanded function and fold space. About a dozen aptamer therapeutic candidates are currently being evaluated in clinical trials. It is to be hoped that MACUGEN will not remain the only clinically approved aptamer for long.
在核酸药物研发领域中，使用适应的聚合酶和异构核酸（XNAs）的 SELEX 技术是最近的一项令人激动的进展。基于 2'-F-阿拉伯核酸（FANA）、（3'→2'）-L-α-噻吕糖核酸（TNA）和其他 XNAs 的寡核苷酸适配体在抵抗降解方面具有高度稳定性，并为扩展功能和结构空间提供了可能性。目前大约有十几个适配体治疗候选药物正在进行临床试验评估，这表明适配体作为治疗药物的潜力与日俱增。希望 MACUGEN 不会长期成为唯一获得临床批准的适配体。
