Lipid-anchored oligonucleotides for stable double-helix formation in distinct membrane domains
Lipid-anchored oligonucleotides for stable double-helix formation in distinct membrane domains
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DOI：
10.1002/anie.200600822
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Herrmann, Andreas
中科院分区：
文献类型：
--
作者：
Kurz, Anke;Bunge, Andreas;Herrmann, Andreas
脂质锚定的RNA和DNA寡核苷酸已经成为有吸引力的嵌合分子，用于纳米生物技术、细胞生物学和医学治疗策略的开发中的各种应用。[1，2]例如，已经合成了亲脂性寡核苷酸以改善反义单链DNA（ssDNA）和干扰RNA的细胞摄取。[1，3]最近，有报道称，注射与胆固醇锚偶联的短干扰RNA分子（siRNA）后，小鼠体内的内源性载脂蛋白B有效沉默。[4]根据应用，脂质锚定的寡核苷酸应满足各种要求，例如容易和灵活的合成，有效和稳定的膜掺入，以及互补DNA链的选择性结合。出于各种目的，期望寡核苷酸在膜中的独特横向组织。通过选择适当的脂质锚，可以实现亲脂性寡核苷酸在特定脂质结构域中的富集[5]，这允许在膜表面上产生不同的功能区室。在此，我们描述了一种脂化的ssDNA分子的合成和应用，该分子稳定地插入脂质膜，在囊泡表面上显示寡核苷酸，通过形成沃森-克里克碱基对结合互补DNA链，并优先螯合到液体无序的膜结构域中。寡核苷酸LT 23聚体是由21个胸苷单元和位置1和8的两个亲脂性核苷酸L组成的23聚体，其中α-生育酚单元作为亲脂性锚（方案1）。使用亚磷酰胺方法（支持信息）在DNA合成仪上合成LT 23聚体。亲脂性生育酚丙酰基胞苷L通过使用最近公布的方法[6]将5-碘胞苷与外消旋O-炔丙基生育酚进行Sonogashira偶联获得，并通过常规方法转化为5 '-DMTr-保护的3'-二异丙基氨基氰基乙基亚磷酰胺。这种方法提供了一个高度的灵活性，因为亲脂性核苷酸可以引入到任何位置的oligonucleotide.荧光显微镜被用来可视化的LT 23聚体的膜亲和力和结合互补的A20聚体DNA链的能力。制备含有1 mol%LT 23 mer和荧光磷脂类似物NNBD-PE（0.5 mol%;参见实验部分）的巨大单层囊泡（GUV）。为了证明互补DNA链的结合，加入在其3 '（3' Rh-A20聚体）或在其5 '末端（5' Rh-A20聚体）用荧光团罗丹明（Rh）标记的腺苷20聚体。这些互补链与GUV膜的LT 23聚体的结合可以通过Rh荧光来鉴定（仅图1
Lipid-anchored RNA and DNA oligonucleotides have emerged as attractive chimeric molecules for various applications in nanobiotechnology, in cell biology, and for the development of therapeutic strategies in medicine.[1, 2] For example, lipophilic oligonucleotides have been synthesized to improve cellular uptake of antisense single-stranded DNA (ssDNA) and interference RNA.[1, 3] Recently, efficient silencing of an endogenous apolipoproteinB in mice has been reported upon injection of short interfering RNA molecules (siRNA) coupled to a cholesterol anchor.[4] Depending on the application, lipid-anchored oligonucleotides should meet various requirements, such as easy and flexible synthesis, efficient and stable membrane incorporation, and selective binding of complementary DNA strands. For various purposes a distinct lateral organization of oligonucleotides in membranes is desirable. By selecting appropriate lipid anchors, the enrichment of lipophilic oligonucleotides in specific lipid domains can be achieved [5] which allows distinct functional compartments to be created on a membrane surface. Herein we describe the synthesis and application of a lipidated ssDNA molecule that stably inserts into lipid membranes, displays the oligonucleotide on the vesicular surface, binds complementary DNA strands by formation of Watson–Crick base pairs, and preferentially sequesters into liquid-disordered membrane domains. The oligonucleotide LT23mer is a 23mer consisting of 21 thymidine units and two lipophilic nucleotides L in positions 1 and 8 with α-tocopherol units as lipophilic anchors (Scheme 1). LT23mer was synthesized on a DNA synthesizer applying phosphoramidite methodology (Supporting Information). The lipophilic tocopherylpropinylcytidine L was obtained by Sonogashira coupling of 5-iodocytidine with racemic O-propargyltocopherol using a recently published procedure [6] and transformed into the 5’-DMTr-protected 3’-diisopropylaminocyanethylphosphor amidite by conventional methods. This method provides a high flexibility because lipophilic nucleotides can be introduced in any position of an oligonucleotide.Fluorescence microscopy was used to visualize the membrane affinity of LT23mer and the ability to bind a complementary A20mer DNA strand. Giant unilamellar vesicles (GUVs) were prepared containing 1 mol% LT23mer and the fluorescent phospholipid analogue NNBD-PE (0.5 mol%; see Experimental Section). To demonstrate binding of the complementary DNA strand, an adenosine 20mer tagged either on its 3’(3’Rh-A20mer) or on its 5’terminus (5’Rh-A20mer) with the fluorophore rhodamine (Rh) was added. The binding of these complementary strands to the LT23mer of the GUV membranes could be identified by the Rh fluorescence (Figure 1, only