CN116426532B - 一种靶向适配体及其应用 - Google Patents
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Abstract
本发明属于生物医药领域,涉及一种靶向适配体及其应用。所述的靶向适配体对肝脏具有很好的特异性和亲和性。所述的靶向适配体具有特异性高、稳定性高、可体外合成、易于修饰、稳定性高等优点,可用于肝脏细胞或组织的检测及靶向递送产品的制备。
Description
技术领域
本发明属于生物医药领域,涉及一种靶向适配体及其应用。
背景技术
核酸适配体(Aptamer)是一类短的结构明确的单链RNA或DNA分子,可以与特定分子靶标进行特异性结合,长度为20-100 nt(常见为20-40 nt)。近年来,由于核酸适配体能与细胞表面受体结合被细胞内吞的特点,使其被应用于靶向药物递送,一般直接或用接头把Aptamer与siRNA、ASO、Gapmer等核酸药物连接在一起行使靶向递送药物的功能。
Aptamer的产生,是通过指数富集的配体***进化,进行迭代富集筛选,简称为“SELEX”(Systematic Evolution of Ligands by Exponential Enrichment)。从1990年至今的研究表明,其大体思路分三步进行:(1)识别结合,(2)分离提取,(3)扩增往复。具体分类按照筛选对象主要分为两大类,体外筛选(in vitro SELEX)和体内筛选(in vivoSELEX);进一步按筛选原材料的不同又可分为细胞富集筛选法、组织富集筛选法和活体富集筛选法。
体外进行的细胞富集筛选方法,是使用细胞作为筛选目标体。其特点是可以使被筛选的备选物以天然完整的形式与细胞表面靶标物质相互作用,无需提前验证知晓具体靶标物。适配体可以与细胞表面的任何潜在物质,如膜蛋白、多糖、脂质等发生互作。细胞富集筛选方法因具体技术手段不同,还可以细分为荧光激活细胞分选筛选法(Fluorescence-activated cell sorting,FACS)、细胞内化筛选法(Cell-Internalized SELEX)、微流控芯片细胞筛选法(On-Chip Cell SELEX)、配体介导细胞筛选法(Ligand-guided cell-SELEX,LGCS)、环套细胞筛选法(Toggle-Cell-SELEX)等等。
利用细胞作为富集筛选工具有别于组织和活体的两大好处,就是实施实验便捷和操作环境稳定。与此同时局限性也很明显,适配体最终的预期使用场景是个体,而往往在细胞环境下表现良好的适配体在组织或者个体中却显示出极低的亲和力,更有甚者转移阵地的适配体以失败告终。为了提高适配体对组织和器官的作用效率,克服组织器官中细胞的复杂性、异质性和形态结构多样性带来的影响,利用整个组织进行富集筛选,是一个更优的手段。此方法常在肿瘤组织中使用组织切片进行富集分析,如乳腺癌和导管癌等。
发明内容
本发明的第一方面提供一种适配体,所述适配体为如SEQ ID NO:1所述的寡核苷酸。
在一些实施方案中,所述适配体可以为人工合成的序列,或任何其他来源的核苷酸序列。
本发明的第二方面提供一种适配体的衍生物,包括A1至A4中的至少一种:
A1:将所述的核酸适配体的一端或中间连接上信号分子和/或活性分子和/或功能基团,且具有相同功能的核酸适配体的衍生物;
A2:由所述的适配体编码和/或转录的RNA;
A3:由所述的适配体编码的肽核酸;
A4:将所述的核酸适配体进行核苷酸修饰,且具有相同功能的适配体的衍生物。
在一些实施方案中,所述信号分子和/或活性分子和/或功能基团包括荧光基团、淬灭基团、放射性物质、蛋白、抗体、siRNA、ASO、Gapmer、氨基、异硫氰酸荧光素、生物素、地高辛、纳米材料、聚乙二醇、叶酸和酶标记中的至少一种。在一些实施方案中,所述荧光基团包括Cy3、Cy5、FAM、荧光素、FITC、荧光纳米微球。
在一些实施方案中,所述核苷酸修饰包括氨基化、磷酸化、羧基化、硫代修饰、巯基修饰、2-甲氧基修饰、2-甲氧基乙基修饰、2-氟修饰、锁核酸修饰、3'端倒置dT/dG修饰、甲基/去甲基化和同位素化的修饰中的至少一种。
在一些实施方案中,所述适配体可以通过一些不改变核苷酸同一性的核酸修饰来修饰。例如,核酸的骨架或糖残基的修饰不改变核苷酸的同一性,因为碱基本身保持与适配体序列中的相同。
本发明的第三方面提供所述的适配体,或所述的适配体衍生物在制备靶向肝脏的制剂中的应用。
在一些实施方案中,所述靶向肝脏的制剂为:
B1:特异性靶向肝脏细胞或者肝脏组织的靶向性药物;
B2:特异性诊断肝脏疾病的诊断试剂;
B3:肝脏细胞、组织或活体定位成像制剂;或
B4:捕获或提取待测样品中的肝脏细胞或组织的制剂。
在一些实施方案中,所述应用还包括非诊断和治疗目的的应用。
本发明的第四方面提供一种靶向递送载体,包括所述的适配体,或所述的适配体的衍生物。
本发明的第五方面提供一种肝脏靶向药物,包括:
a)所述的适配体,或所述的适配体的衍生物;和
b)与a)直接或间接连接的药物。
本发明的第六方面提供一种肝脏细胞检测试剂,包括:
i)所述的适配体,或所述的适配体的衍生物;和
ii)与i)直接或间接连接的可检测标记。
在一些实施方案中,所述“间接连接”可以通过,例如,通过配体(ligand)或接头(linker)连接。
在一些实施方案中,所述可检测的标记为荧光基团。在一些实施方案中,所述荧光基团包括Cy3、Cy5、FAM、荧光素、FITC、荧光纳米微球。
本发明的第七方面提供一种在体外向受试细胞或组织递送靶向药物的方法,包括:
i)将所述的适配体,或所述的适配体的衍生物,或所述的靶向递送载体,和靶向药物作用于受试细胞或组织;或
ii)将所述的肝脏靶向药物作用于受试细胞或组织;
所述方法用于非疾病的诊断与治疗。
本发明的第八方面提供一种靶向递送***,包括:
i) 靶向递送构件;和
ii) 治疗/诊断构件;
所述靶向递送构件包括所述的适配体,或所述的适配体的衍生物,或所述的靶向递送载体。
附图说明
图1为小鼠体内筛选肝脏靶向适配体序列流程;包括待筛选的Aptamer序列制备及体外转录成修饰的RNA,注射到小鼠体内后取肝脏组织进行RNA提取,得到的RNA一部分用于逆转录建库测序分析不同Aptamer在组织中的富集程度,另一部分用于逆转录加接头进行体外转录做第二轮筛选。
图2为Aptamer在两轮筛选中的富集度;富集度的计算方法为,该条Aptamer在测序数据中被测到的读长数除以所有Aptamer测到的读长数的和。
图3为Aptamer1与小鼠肝原代细胞孵育15min结果;左图为cy3荧光通道拍摄,右图为紫外通道拍摄。
图4为Aptamer1转染2h后肝原代细胞荧光图;拍摄通道为cy3荧光通道。
图5为Aptamer25(第1行)和Aptamer26(第2行)与小鼠肝原代细胞孵育24h结果;左图为cy3荧光通道拍摄,右图为紫外通道拍摄。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案,具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
如本发明所用:
术语:RNA骨架修饰:硫代磷酸化;
RNA的碱基修饰:2'-氧甲基修饰(2'-O-Me):在碱基的2'位加上一个甲基基团;2'-氟修饰(2'-F):在碱基的2'位用氟原子替代氢原子;锁核酸修饰(locked nucleic acid,LNA):核酸糖环被氧原子连接到5'-碳和2'-碳之间;2'-MOE:在碱基的2'位引入2'-O-甲氧基乙基。
术语“靶向递送***”是指能够将活性成分递送至靶部位的***,即能够靶向递送,优选地能够在患者体内靶向递送。
术语“靶向递送”是指将治疗剂或诊断剂(例如,药物、检测试剂)递送至对象(例如患者),特别是递送至患者体内的细胞中。靶向递送还包括“靶向治疗递送”,意指同时递送治疗剂和诊断剂,优选递送至患病部位,从而允许同时进行治疗和诊断和/或治疗监测。
术语“序列同一性”是指如通过比较序列而确定的、两个以上多肽分子或两个以上核酸分子的序列之间的关系。在本领域中,“同一性”也意指核酸分子之间或多肽之间的序列相关性程度,根据情况可以由两个以上核苷酸残基或两个以上氨基酸残基之间的匹配数量而确定。
实施例1 在小鼠体内筛选可靶向肝脏的核酸适配体序列
本实施例以小鼠为筛选载体,把待筛选的核酸适配体序列文库皮下注射到小鼠体内并取肝脏组织进行RNA提取及二代测序分析,得到富集的核酸适配体序列再重复此流程进行第二轮筛选,具体筛选流程如图1所示。
具体的实验流程如下:
1.首轮筛选文库制备
(1)文库合成:待筛选序列文库由5’端T7启动子(斜体),加上两端恒定序列(单下划线)和中间一段长度为40个碱基的Aptamer序列(合成时为40个N碱基,N为A,T,C,G中任一碱基)构成,总长为103 nt,按照常规序列合成进行文库合成(示例性的序列信息如表2所示)。首轮筛选的文库包含约440条Aptamer序列(未全部示出)。
(2)体外转录:使用lucigen公司的T7转录试剂盒,按照说明书的方法进行体外转录,得到碱基C和U带有2’F修饰的RNA。
(3)RNA纯化:在步骤2中得到的RNA溶液中加入2.1 μL 3M的醋酸钠和52.5 μL的乙醇,置于-20℃孵育30 min。而后放置于4℃低温离心机中12000 rpm离心15 min,去上清液,以70%乙醇轻柔洗涤沉淀物两次,吸净上清液体,加入10 μL 纯水溶解,测浓度,标记备用。
(4)退火:考虑到RNA具有丰富的二级结构,在每次使用前,需将RNA退火处理。条件如下,70°C加热 3分钟,以每分钟降低1℃的降温速率缓慢降温至25℃,保持此温度3分钟。
2.小鼠体内筛选
选择6-8周龄、20-30g的雌性C57BL/6小鼠,经检查无明显异常,进行适应性饲养5天后,选取无异常小鼠开展实验。
将10 μg修饰的RNA使用PBS稀释至100 μL(注射材料),吸入无菌1 mL注射器内,排净空气,放置至室温备用。
具体流程:
(1)皮下注射:将注射材料吸入无菌1mL注射器,固定好小鼠,使用酒精对小鼠脖颈注射处进行消毒,将注射材料缓慢推入小鼠皮下,轻轻揉动注射处,使之吸收,计时30-60分钟。
(2)取材步骤:将小鼠固定稳妥后,通过腹腔注射3%戊巴比妥钠溶液(1.5 mL/kg)进行全身麻醉。
(3)心脏灌流:腹部备皮,涂抹酒精消毒,手术剪快速开腹,心脏灌流后剪出所需肝脏组织,将之迅速置于加有1mL TRIzol的研磨管中,盖紧放入液氮速冻待用。
样品处理:将所取组织放入已降温完成的Precellys Evolution均质器内充分研磨,使用酚氯仿法提取RNA。
3.高通量测序获取肝脏中不同Aptamer的丰度
上步骤中获得的RNA用特异性的逆转录引物对RNA进行逆转录反应得到cDNA,再以cDNA作为模板,依次使用第一轮(round1)和第二轮(round2)的扩增引物对cDNA进行扩增并最终获得总长度为200bp左右的待测高通量测序文库,引物序列如表1所示。
表1.引物序列表
引物名称 | 引物序列 |
逆转录引物 | TCTCGGATCCTCAGCGAGTG |
扩增引物-F | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTG |
扩增引物-R | TCTCGGATCCTCAGCGAGTG |
Round1-F | CGACGCTCTTCCGATCTGGGCGTCAGTCGTCTG |
Round1-R | GTGTGCTCTTCCGATCTTCTCGGATCCTCAGCGAGTG |
Round2-F | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT |
Round2-R | CAAGCAGAAGACGGCATACGAGATATCACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT |
4.高通量测序及数据分析
建好的高通量测序文库交由安升达公司在HiSeq X Ten(Illumina)仪器上进行高通量测序,得到原始测序数据后进行数据处理,并分析每种Aptamer序列测到的读长数及其富集度,富集度的计算方法为每条Aptamer测序获得的读长数除以所有Aptamer测序获得的总读长数得到的比例。
5. 第二轮筛选文库制备
(1)组织研磨及RNA提取:将收集得到的液氮速冻后的组织进行研磨及常规RNA提取,制得本轮获得的总RNA。
(2)反转录:使用步骤(1)中所得总RNA用于反转录。其中使用的引物为表1中的逆转录引物。
(3)连接T7启动子:反转后的cDNA利用两端恒定序列通过表1中的扩增引物将T7启动子序列(同表1)通过PCR的方法连接至5’端,便于实施体外转录。
(4)体外转录:此处转录反应条件与“1.首轮筛选文库制备”中一致,把(3)中得到的双链DNA用于此步骤的体外转录。
6. 第二轮小鼠体内筛选
第二轮小鼠体内筛选流程与第一轮小鼠体内筛选方法一致,把RNA通过皮下注射到小鼠体内后,对小鼠进行麻醉及心脏灌注,取肝脏组织进行RNA提取,及高通量建库测序。
以筛选出的26条Aptamer序列为例(示例如表2,其中未划线的为Aptamer序列,划线的为第一轮筛选文库制备步骤中的添加的序列(斜体为5’端T7启动子,单下划线为两端恒定序列))。
表2. 示例性Aptamer序列信息(含第一轮筛选文库制备步骤中的启动子和两端恒定序列)
序列名 | 序列信息(5’-3’) | SEQ ID NO: |
Seq1 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCGGGCTCTAGCTTGACTAGTTCCCTTAGACCTTCTGAGGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:27 |
Seq2 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCATACCAGTCTGCACGATTGGTCTGTGGACCGACTCGGCGCACTCGCTGAGGATCCGAGA | SEQ ID NO:28 |
Seq3 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGTATTCTGGCACTTAAAGTGTTCACGGATGTAGAGGTGGCGCACTCGCTGAGGATCCGAGA | SEQ ID NO:29 |
Seq4 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCGCTTTGTGTGGTACACAGAGCGGGATGGCCCTACGACGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:30 |
Seq5 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGGGTTTGACTTGCCGTCATTCCTGCAGTGACTTGGGGCGACACTCGCTGAGGATCCGAGA | SEQ ID NO:31 |
Seq6 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGCCTGGCAAACTTCGCTTTGCCGCTCGATTCCTGAGGCACCACTCGCTGAGGATCCGAGA | SEQ ID NO:32 |
Seq7 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGTGTTCACATTCACTTGTGAGGACCCTTCGATCCTTCGCCCACTCGCTGAGGATCCGAGA | SEQ ID NO:33 |
Seq8 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCCCCGGTTCGTTGCCCAACGTGCCGGTTGCTGATGGCTAGCACTCGCTGAGGATCCGAGA | SEQ ID NO:34 |
Seq9 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCCAGATATCGGACACGATAGTCTGGCGCTTCCGATCGACGCACTCGCTGAGGATCCGAGA | SEQ ID NO:35 |
Seq10 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCCGCGGGAGGGCAAACCTCCTGCGTTGCTCGACCGCATGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:36 |
Seq11 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGAGAGTAGCACGAGTTGCTATTCGTCTCGTCGTTTGGTGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:37 |
Seq12 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCCATTAACGGGCTTACAGTTCGTTCTTGGTCAGGGATGGACACTCGCTGAGGATCCGAGA | SEQ ID NO:38 |
Seq13 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGTCCGATAAGTTTCGCACTTACGGTAGCCATCGTCTGGGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:39 |
Seq14 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCTCGCGGCGAAGCTGTGATGTAGCCGACTCGGCGGCACGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:40 |
Seq15 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCGTCTCCGTTTCACCGGGGGCGCGTTCGTTCCTGACGCGACACTCGCTGAGGATCCGAGA | SEQ ID NO:41 |
Seq16 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGACCTGCGAACCGCTTCGTGGGTGGACCTAACGCCCCGGTGCACTCGCTGAGGATCCGAGA | SEQ ID NO:42 |
Seq17 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCTCTTACCATCTAAGCGATGGAAACGCTGCTGGCGAGACGCACTCGCTGAGGATCCGAGA | SEQ ID NO:43 |
Seq18 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGCCCAGGCGACGTCCTGGTTTCGCTGGGCGATTAACGCGACACTCGCTGAGGATCCGAGA | SEQ ID NO:44 |
Seq19 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGGACACTGTCACAACGACGGGAACTGGAGTGACTTTGGCGCACTCGCTGAGGATCCGAGA | SEQ ID NO:45 |
Seq20 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCTGGCAATGCGAAATGCTTGTCCTTCAGAGGCCTGGCGCGCACTCGCTGAGGATCCGAGA | SEQ ID NO:46 |
Seq21 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGTTTCCGTGCGCTAATGCGTTGTCGGAGTGGACCTGTTCGTCACTCGCTGAGGATCCGAGA | SEQ ID NO:47 |
Seq22 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCAATTGCGCCGTGAGGTGTAGTCGCACGATGGATTGGGTGCACTCGCTGAGGATCCGAGA | SEQ ID NO:48 |
Seq23 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGCACTCTACGGGTGTTGCTTCTGACCTCCCACCTTGACAGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:49 |
Seq24 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGGGACTTGCCCGTTCCGCGCGCTGTCGTCTACCCGGATGGGCACTCGCTGAGGATCCGAGA | SEQ ID NO:50 |
Seq25 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGTAACCTGCTCGGGAACGAAAAGGTCATGGGTCGCGGGCGCCACTCGCTGAGGATCCGAGA | SEQ ID NO:51 |
Seq26 | GGGGGAATTCTAATACGACTCACTATAGGGCGTCAGTCGTCTGTCTTCCCGTTTACAGAACGGGGGCTTCTGCGCCTGCGCCACACTCGCTGAGGATCCGAGA | SEQ ID NO:52 |
根据高通量建库测序结果,分析每种Aptamer的测序读长及富集度。
富集度的计算方式为:每条Aptamer测序获得的读长数除以所有参比Aptamer测序获得的总读长数得到的比例。
示例性文库序列信息所示的Aptamer对应的富集度分析结果如表3所示。
结果表明,Aptamer1在所有Aptamer中的占比从第一轮的17.25%提升到第二轮的26.41%,如图2及表3所示。除Aptamer1的显著富集外,Aptamer2也从第一轮的7.73%提升到13.91%以及Aptamer9从1.31%提升到2.91%。同时,也有一些Aptamer从第一轮的富集度高到第二轮的富集度变低,说明该Aptamer并不是肝脏中最适的Aptamer,例如Aptamer7从第一轮的15.26%降低到第二轮的3.55%,Aptamer10从第一轮的6.07%降低到2.79%。
表3. 示例性的Aptamer在两轮筛选中的富集度
序列名 | 序列信息 | 第二轮测序读长数 | 第二轮占比(%) | 第一轮测序读长数 | 第一轮占比(%) |
Aptamer1(SEQ ID NO:1) | CGGGCUCUAGCUUGACUAGUUCCCUUAGACCUUCUGAGGC | 73721 | 26.41 | 27006 | 17.25 |
Aptamer2(SEQ ID NO:2) | CAUACCAGUCUGCACGAUUGGUCUGUGGACCGACUCGGCG | 38829 | 13.91 | 12096 | 7.73 |
Aptamer3(SEQ ID NO:3) | UAUUCUGGCACUUAAAGUGUUCACGGAUGUAGAGGUGGCG | 16297 | 5.84 | 6034 | 3.86 |
Aptamer4(SEQ ID NO:4) | CGCUUUGUGUGGUACACAGAGCGGGAUGGCCCUACGACGC | 16073 | 5.76 | 9296 | 5.94 |
Aptamer5(SEQ ID NO:5) | GGGUUUGACUUGCCGUCAUUCCUGCAGUGACUUGGGGCGA | 13889 | 4.98 | 6346 | 4.05 |
Aptamer6(SEQ ID NO:6) | GCCUGGCAAACUUCGCUUUGCCGCUCGAUUCCUGAGGCAC | 11423 | 4.09 | 6427 | 4.11 |
Aptamer7(SEQ ID NO:7) | GUGUUCACAUUCACUUGUGAGGACCCUUCGAUCCUUCGCC | 9898 | 3.55 | 23888 | 15.26 |
Aptamer8(SEQ ID NO:8) | CCCCGGUUCGUUGCCCAACGUGCCGGUUGCUGAUGGCUAG | 8906 | 3.19 | 7331 | 4.68 |
Aptamer9(SEQ ID NO:9) | CCAGAUAUCGGACACGAUAGUCUGGCGCUUCCGAUCGACG | 8117 | 2.91 | 2048 | 1.31 |
Aptamer10(SEQ ID NO:10) | CCGCGGGAGGGCAAACCUCCUGCGUUGCUCGACCGCAUGC | 7792 | 2.79 | 9494 | 6.07 |
Aptamer11(SEQ ID NO:11) | GAGAGUAGCACGAGUUGCUAUUCGUCUCGUCGUUUGGUGC | 7513 | 2.69 | 1017 | 0.65 |
Aptamer12(SEQ ID NO:12) | CCAUUAACGGGCUUACAGUUCGUUCUUGGUCAGGGAUGGA | 7253 | 2.60 | 7023 | 4.49 |
Aptamer13(SEQ ID NO:13) | GUCCGAUAAGUUUCGCACUUACGGUAGCCAUCGUCUGGGC | 7099 | 2.54 | 6581 | 4.20 |
Aptamer14(SEQ ID NO:14) | CUCGCGGCGAAGCUGUGAUGUAGCCGACUCGGCGGCACGC | 5739 | 2.06 | 5855 | 3.74 |
Aptamer15(SEQ ID NO:15) | CGUCUCCGUUUCACCGGGGGCGCGUUCGUUCCUGACGCGA | 4753 | 1.70 | 2891 | 1.85 |
Aptamer16(SEQ ID NO:16) | ACCUGCGAACCGCUUCGUGGGUGGACCUAACGCCCCGGUG | 4742 | 1.70 | 4387 | 2.80 |
Aptamer17(SEQ ID NO:17) | CUCUUACCAUCUAAGCGAUGGAAACGCUGCUGGCGAGACG | 4330 | 1.55 | 5066 | 3.24 |
Aptamer18(SEQ ID NO:18) | GCCCAGGCGACGUCCUGGUUUCGCUGGGCGAUUAACGCGA | 4034 | 1.45 | 4604 | 2.94 |
Aptamer19(SEQ ID NO:19) | GGACACUGUCACAACGACGGGAACUGGAGUGACUUUGGCG | 3939 | 1.41 | 1462 | 0.93 |
Aptamer20(SEQ ID NO:20) | CUGGCAAUGCGAAAUGCUUGUCCUUCAGAGGCCUGGCGCG | 3847 | 1.38 | 218 | 0.14 |
Aptamer21(SEQ ID NO:21) | UUUCCGUGCGCUAAUGCGUUGUCGGAGUGGACCUGUUCGU | 3838 | 1.37 | 85 | 0.05 |
Aptamer22(SEQ ID NO:22) | CAAUUGCGCCGUGAGGUGUAGUCGCACGAUGGAUUGGGUG | 3681 | 1.32 | 206 | 0.13 |
Aptamer23(SEQ ID NO:23) | CACUCUACGGGUGUUGCUUCUGACCUCCCACCUUGACAGC | 3660 | 1.31 | 4426 | 2.83 |
Aptamer24(SEQ ID NO:24) | GGACUUGCCCGUUCCGCGCGCUGUCGUCUACCCGGAUGGG | 3352 | 1.20 | 764 | 0.49 |
Aptamer25(SEQ ID NO:25) | UAACCUGCUCGGGAACGAAAAGGUCAUGGGUCGCGGGCGC | 3313 | 1.19 | 486 | 0.31 |
Aptamer26(SEQ ID NO:26) | UCUUCCCGUUUACAGAACGGGGGCUUCUGCGCCUGCGCCA | 3097 | 1.11 | 1479 | 0.94 |
实施例2 在小鼠原代肝细胞上验证Aptamer序列的递送效果
以富集程度突出的Aptamer1验证其细胞递送效果。
为了进一步验证其进入肝脏细胞的效果,将新鲜分离的小鼠肝原代细胞种于12孔板中,以8 nmol的量将带有荧光基团的Aptamer1(cy3-CGGGCUCUAGCUUGACUAGUUCCCUUAGACCUUCUGAGGC)直接加入细胞培养基中进行观察。
结果表明,在加入Aptamer1约15分钟左右即可观察到明显的荧光,如图3所示,其中左边为荧光通道拍摄的图片,右边为细胞核染色拍摄的图片,几乎百分之百的细胞均进入了大量的筛选出的适配体序列。
核酸药物通过皮下或者静脉注射进入体内则会在血液中循环并最终到达靶器官,而血液中会有大量的核酸酶,核酸药物在血液中循环的时间越久越容易被降解,故其进入细胞的速度会是影响其药代的重要因素。
通过把Aptamer1的序列直接孵育可知,在15分钟左右时长就可大量进入细胞。
实施例3 商业化脂质体细胞转染的递送效果对比
细胞转染是通过商业化的脂质体包裹DNA或RNA序列并把其递送到细胞内的方式。按照Lipofectamine RNAiMAX Transfection Reagent(赛默飞公司)说明书的操作指引,直接把转染试剂与RNA进行混合室温孵育5分钟后加入细胞中,通过比较二者进入细胞的速度可评估Aptamer递送的效果。
结果表明,转染组直到两小时才可见细胞膜周围的荧光出现(如图4所示),通过此对比可知本发明获得的Aptamer1序列是一条可以快速进入细胞的核酸适配体序列。
实施例4 不同富集程度的Aptamer的递送效果
同时对:
Aptamer25(cy3-UAACCUGCUCGGGAACGAAAAGGUCAUGGGUCGCGGGCGC)和Aptamer26(cy3-UCUUCCCGUUUACAGAACGGGGGCUUCUGCGCCUGCGCCA)进行小鼠原代肝细胞孵育验证(实验步骤同实施例2)。结果表明Aptamer25、Aptamer26实验组在约24h实验细胞出现比较稳定明显的荧光信号(图5),明显慢于Aptamer1,但仍快于商业化的脂质体。
Claims (8)
1.一种适配体,其特征在于,所述适配体为如SEQ ID NO:1所示的寡核苷酸。
2.如权利要求1所述的适配体的衍生物,其特征在于,包括A1和A4中的至少一种:
A1:将权利要求1所述的核酸适配体的一端或中间连接上信号分子和/或活性分子和/或功能基团,所述信号分子和/或活性分子和/或功能基团包括荧光基团、淬灭基团、放射性物质、蛋白、siRNA、ASO、Gapmer、异硫氰酸荧光素、生物素、地高辛、纳米材料、聚乙二醇、叶酸中的至少一种,且具有相同功能的核酸适配体的衍生物;
A4:将权利要求1所述的核酸适配体进行核苷酸修饰,所述核苷酸修饰包括氨基化、磷酸化、羧基化、硫代修饰、巯基修饰、2-甲氧基修饰、2-甲氧基乙基修饰、2-氟修饰、锁核酸修饰、3'端倒置dT/dG修饰、甲基/去甲基化和同位素化的修饰中的至少一种,且具有相同功能的适配体的衍生物。
3.如权利要求1所述的适配体,或权利要求2所述的适配体的衍生物在制备靶向肝脏的制剂中的应用。
4.如权利要求3所述的应用,其特征在于,所述的靶向肝脏的制剂为:
B1:特异性靶向肝脏细胞或者肝脏组织的靶向性药物;
B2:特异性诊断肝脏疾病的诊断试剂;
B3:肝脏细胞、组织或活体定位成像制剂;或
B4:捕获或提取待测样品中的肝脏细胞或组织的制剂。
5.一种靶向递送载体,其特征在于,包括如权利要求1所述的适配体,或权利要求2所述的适配体的衍生物。
6.一种肝脏靶向药物,其特征在于,包括:
a) 如权利要求1所述的适配体,或权利要求2所述的适配体的衍生物;和
b)与a)直接或间接连接的药物。
7.一种肝脏细胞检测试剂,其特征在于,包括:
i)如权利要求1所述的适配体,或权利要求2所述的适配体的衍生物;和
ii)与i)直接或间接连接的可检测标记。
8.一种在体外向受试细胞或组织靶向递送药物的方法,其特征在于,包括:
i)将权利要求1所述的适配体,或权利要求2所述的适配体的衍生物,或权利要求5所述的靶向递送载体,和药物直接或间接连接,靶向作用于受试细胞或组织;或
ii)将权利要求6所述的肝脏靶向药物作用于受试细胞或组织;
所述方法用于非疾病的诊断与治疗。
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