CN116445479A - 工程化DNAzyme分子机器及其应用 - Google Patents
工程化DNAzyme分子机器及其应用 Download PDFInfo
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Abstract
本发明提供工程化DNAzyme分子机器及其应用,包括链组一和链组二中的一组或两组,链组一:i‑motif链修饰的DNAzyme及其底物链,所述链组一修饰在癌细胞与T细胞表面,利用肿瘤细胞的酸性微环境使其修饰的i‑motif链收缩,缩短癌细胞与肿瘤细胞间距离;链组二:线粒体靶向肽修饰的DNAzyme及其底物链,利用细胞内线粒体聚集来诱导细胞凋亡。基于DNAzyme的肿瘤微环境响应性控制细胞外和细胞内的细胞行为,包括细胞识别、细胞间接近、治疗后分离和线粒体聚集,不仅为调节细胞间相互作用提供了有用的途径,同时也提供了一种强大的具有针对性的癌症治疗方法,而对正常细胞不会产生杀伤。
Description
技术领域
本发明涉及细胞活体成像和纳米材料技术领域,尤其涉及用于癌症治疗的工程化DNAzyme分子机器。
背景技术
癌症是世界上一个重要的死亡原因,许多人都致力于开发更好的癌症治疗方法。侵袭性肿瘤的情况尤其严重,因为它们快速增殖并促进肿瘤恶性化。为提高此类病例的治疗效果,典型的策略是使用大剂量的非特异性化疗药物,如阿霉素、紫杉醇、甲氨蝶呤等。然而,此类药物的广泛使用可能会对正常细胞造成损害,从而导致严重的副作用。为了减少副作用,同时保持癌症治疗效果,联合疗法提供了一种将不同的单一疗法结合在一起的方法。一种典型的联合治疗策略是将免疫治疗与纳米颗粒作为药物载体相结合,同时通过纳米颗粒给药提高细胞内药物浓度,从而诱导对癌细胞的免疫杀伤。然而,大多数报道的联合治疗方法都集中于通过使用大量IgG抗体诱导T细胞和癌细胞之间的识别,但是这种大分子可能会影响免疫反应并阻碍药物进入癌细胞。因此,尽管在这一领域取得了进展,但设计一种简单的策略来实现T细胞和癌细胞之间的细胞-细胞相互作用的动态操纵仍然具有挑战性,包括细胞识别、细胞-细胞接近和治疗后分离。此外,对于细胞内治疗的一部分,重要的是减少药物对正常细胞的副作用。
第一个DNAzyme于1994年被报道用于RNA切割。从那时起,许多不同类型的DNAzymes被报道,催化RNA/DNA裂解、连接、磷酸化和其他反应。由于DNAzymes是通过体外选择获得的具有催化活性的DNA分子,因此具有rna切割位点的DNAzymes由于其出色的可编程性、稳定性和活性,在治疗和诊断应用方面都受到了极大的关注。许多基于寡核苷酸的生物制药已经被用于基因治疗,如反义寡核苷酸、小干扰rna(sirna)、核酶和dna酶。与核酶相比,DNAzymes具有更高的成本效益、更容易合成、更稳定、更容易标记等优点,为此类应用提供了更好的选择。到目前为止,DNAzymes已经显示出作为多种疾病治疗药物的潜力,如抗病毒、抗菌、抗癌、抗炎,以及动脉粥样硬化。DNAzyme最初用于体外检测HIV RNA切割。DNAzyme也被设计用于治疗虽然有许多有前途的应用报告,但FDA批准的dnazyme为基础的药物尚未进入市场。这表明在这一领域存在一些挑战。与其他基因沉默技术相比,DNAzyme还没有达到相同的发展阶段。
发明内容
本发明的第一个目的在于,提供一种基于工程化DNAzyme分子机器的联合治疗策略,通过细胞外对T细胞/癌细胞相互作用的控制和细胞内对线粒体聚集的控制来诱导癌细胞凋亡。
本发明的第二个目的在于,提供工程化DNAzyme分子机器在制备抗肿瘤药物中的应用。
为了实现上述目的,本发明提供了工程化DNAzyme分子机器,包括链组一和链组二中的一组或两组,
链组一:i-motif链修饰的DNAzyme及其底物链,所述链组一修饰在癌细胞与T细胞表面,利用肿瘤细胞的酸性微环境使其修饰的i-motif链收缩,缩短癌细胞与肿瘤细胞间距离;
链组二:线粒体靶向肽修饰的DNAzyme及其底物链,利用细胞内线粒体聚集来诱导细胞凋亡。
作为一个优选方案,所述链组一包括三条DNA链,其中链1由长脂链、i-motif链和DNAzyme链组成,链2由识别肿瘤细胞高表达的蛋白适配体和DNAzyme相对应的底物链组成,链3由i-motif的互补链和长脂链组成。
连接的i-motif链包括一条或多条,通过修饰个数不同实现不同距离调控,使T细胞能够更高效的进行肿瘤细胞的杀伤。
作为一个优选方案,所述链1和链2还包括pH不敏感的荧光基团,所述链3还包括猝灭剂。
作为一个优选方案,所述荧光基团包括TAMRA、AF 488、CY3、AF 555、AF 532或AF546中的一种。
作为一个优选方案,所述长脂链包括胆固醇、C6 Spacer、C12 Spacer或C18Spacer中的一种。
作为一个优选方案,所述识别肿瘤细胞高表达的蛋白适配体包括MUC1适配体、PD-L1适配体或EGFR适配体中的一种。
作为一个优选方案,所述链组一的三条DNA链分别为:
链1:Cholesteryl-CCCTAACCCTAACCCTAACCCATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA;(SEQ ID NO.1)
链2:GCAGTTGATCCTTTGGATACCCTGGTTGCAGGTAGAGAAGTrAGGAAACTAT;(SEQ IDNO.2)
链3:GTT AGTGTTAGTGTT AG-Cholesteryl(SEQ ID NO.3)。
作为一个优选方案,所述链组二包括两条DNA链,其中链4由线粒体靶向肽和DNAzyme链组成,链5由线粒体靶向肽和DNAzyme链对应的底物链组成。
作为一个优选方案,所述链4和链5还包括荧光基团。
作为一个优选方案,所述线粒体靶向肽包括R8:NH2-(Arg)8-CONH2或Fmoc-FFFGKsuccG-COOH中的一种。
作为一个优选方案,所述链组二的两条DNA链分别为:
链4:R8-ATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA;(SEQ ID NO.4)
链5:R8-TTGCAGGTAGAGAAGTrAGGAAACTAT(SEQ ID NO.5)。
本发明中i-motif链是指是一种特殊的DNA二级结构,它是由四个胞嘧啶重复序列在质子的参与下形成的四链螺旋,该结构只有在酸性环境才能维持。在酸性条件下,富含与鸟嘌呤互补的胞嘧啶碱基C的DNA分子可以通过胞嘧啶C碱基的半质子化C.C+碱基对形成稳定的平行双螺旋,两个平行双螺旋上的C.C+碱基对可以以交替排列和互相嵌入的形式形成四螺旋结构。它的序列为,常用的比如5’-CCC TAA CCC TAA CCC TAA CCC T-3’(SEQ IDNO.6),5’-TCCCTAACCCTAACCCTAACCCAA-3’(SEQ ID NO.7)(Hongbo Chen,Hongxia Sun,etal.Chelerythrine as a fluorescent light-up ligand for an i-motif DNAstructure,New J.Chem.,2021,45,28)。
i-motif的互补链是指可以与i-motif形成双链的DNA序列。常用的比如为:5’-GTTAGT GTT AGT GTT AG-3’(SEQ ID NO.8),或者5’-GGA TTC GCC TTT CGC TTA-3’(SEQ IDNO.9)。
DNAzyme是指一类具有催化功能的DNA分子。同蛋白质和RNA催化酶一样,DNAzyme能够催化多种类型的生化反应。常用的序列如:5’-ATA GTT TCT CCG AGC CGG TCG AAACTT CTC TAC CTG CAA-3’(SEQ ID NO.10),或者5’-ACA GAC ATC TCT TCT CCG AGC CGGCTG AAA TAG TGA GT-3’(SEQ ID NO.11)。
DNAzyme底物链是指与DNAzyme链能够特异性结合从而在特定金属离子存在下能够解离的DNA序列。常用的序列如:5’-TTG CAG GTA GAG AAG T/rA/G GAA ACT AT-3’(SEQID NO.12),或者5’-ACT CAC TAT/rA/GGA AGA GAT GTC TGT-3’(SEQ ID NO.13)。
本发明中TAMRA、AF 488、CY3、AF 555、AF 532、AF546是指所修饰的荧光基团,都具有pH不敏感的特性,能够用于表征DNA链被修饰于细胞膜上。
胆固醇:一种环戊烷多氢菲的衍生物,化学式为C27H46O,可通过疏水作用插入到细胞膜上。
C6 Spacer、C12 Spacer、C18 Spacer:不同链长的长脂链,可通过疏水作用插入到细胞膜上。
MUC1适配体、PD-L1适配体、EGFR适配体是指通过SELEX技术筛选出能与癌细胞表面高表达的MUC1蛋白,PD-L1蛋白或者EGFR蛋白具有高度亲和性,能够特异性结合该蛋白的核酸序列。
本发明提供了所述工程化DNAzyme分子机器在制备抗肿瘤药物中的应用。肿瘤细胞包括所有锌缺乏的细胞,例如PANC-1,PC12等。一方面,T细胞和癌细胞之间的细胞-细胞相互作用对研究人员特别有吸引力,因为T细胞对癌细胞的特异性识别对于建立有效的免疫治疗非常重要。另一方面,线粒体在癌细胞中的分散增加了癌细胞的侵袭性,线粒体的聚集有利于通过产生过多的活性氧(ROS)导致癌细胞死亡。DNAzyme具有在特定金属离子存在下催化核糖核苷酸断裂的能力。根据DNAzyme对金属依赖性断裂的敏感性,我们以前的工作使用金属离子特异性RNA切割DNAzyme及其各自的底物链作为构建块,设计不同的控制开关来操纵细胞行为,包括单个细胞和多细胞球体的细胞间结合和解聚。在此基础上,在这项工作中,我们通过使用不同功能标签修饰的RNA切割DNAzyme构建分子机器,从而允许细胞或线粒体之间的特定相互作用,以获得更有效的癌症联合治疗选择。
本发明通过细胞外对T细胞/癌细胞相互作用的控制或细胞内对线粒体聚集的控制来诱导癌细胞凋亡,或者将两种内外细胞间调控进行结合以获得更有效的癌症联合治疗方法。在外部调节方面,设计了一组含有适配体和i-motif序列的DNAzyme分子机器来感知和响应酸性肿瘤微环境。使用肿瘤标记物作为目标蛋白,对应的适配体序列可对癌细胞进行特异性识别。酸性微环境触发i-motif序列的折叠,缩短细胞间距离以诱导对癌细胞的杀伤。随后,T细胞可以通过金属离子激活的DNAzyme裂解释放。在内部控制方面,通过癌细胞线粒体聚集,进一步诱导癌细胞凋亡,将含有线粒体靶向肽的DNAzyme分子机器导入癌细胞,以诱导线粒体聚集,产生过量的毒性ROS。以PANC-1细胞为例,我们首次证明,已建立的联合疗法对癌症治疗显示出更好的治疗效果。
本发明的优点在于,基于DNAzyme的肿瘤微环境响应性控制细胞外和细胞内的细胞行为,包括细胞识别、细胞间接近、治疗后分离和线粒体聚集,不仅为调节细胞间相互作用提供了有用的途径,同时也提供了一种强大的具有针对性的癌症治疗方法,而对正常细胞不会产生杀伤。
附图说明
图1为通过基于工程DNAzyme分子机器的肿瘤联合治疗实现T细胞/癌细胞相互作用和线粒体聚集的动态控制的工作原理图。
图2为(a)由链1、链2和链3构建的DNAzyme分子机器对t细胞/癌细胞相互作用的外部调控。步骤1-5:muc1的识别,t细胞与癌细胞的组装,细胞与细胞的接近,t细胞与癌细胞的距离扩大,治疗后的分离。(b)细胞图像显示了(a)中步骤1-5所对应的t细胞与癌细胞之间细胞间相互作用的动态操纵。(c)通过pH调节t细胞与癌细胞之间的距离控制。(d)细胞图像显示不同pH值下细胞-细胞距离的变化。
图3为(a)Zn2+特异性DNAzyme控制细胞外线粒体聚集和离解(1组:链4修饰,2组:链5修饰)。(b)细胞图像显示受控的1组和2组细胞外线粒体相互作用(1:1)。(c)细胞外线粒体的TEM图像。(d)DNAzyme分子机器(链4和链5,脂质体传递)对细胞内线粒体相互作用的内部调节。(e)荧光图像显示PANC-1细胞经含链4和链5的脂质体处理1小时,然后在不同时间观察。(f)三维细胞图像显示组装的线粒体。(g)细胞内线粒体的TEM图像。
图4为将链1,链3上的胆固醇替换成C6,C12的长脂链后仍可实现细胞识别、聚集。
图5为将链1上的imotif链变成2到3个后可实现不同距离的细胞间调控。
图6为(a)DNAzyme分子机器杀死联合癌细胞。从(i)至(vi):(i)对照,(ii)PANC-1细胞与未修饰的t细胞混合,(iii)链-2修饰的PANC-1细胞与链-1/链-3修饰的t细胞混合(DNAzyme分子机器用于外部控制t细胞/癌细胞相互作用),(v)PANC-1细胞与脂质体包裹的链-4/链-5混合(DNAzyme分子机器用于线粒体聚集的内部控制),(v)PANC-1细胞与未修饰的t细胞和脂质体包裹的链-4/链-5混合,(vi)链-2修饰的PANC-1细胞与链-1/链-3修饰的t细胞和脂质体包裹的链-4/链-5细胞(联合治疗)。(b)细胞凋亡的荧光图像,图像显示了PANC-1细胞用不同的方法治疗1h,相应对异物(i)在(a)(vi)。(c)通道强度。(d)荧光强度比较直方图。(e)插图显示联合治疗过程。
具体实施方式
下面结合具体实施例,进一步阐述本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1.T细胞和癌细胞之间细胞-细胞相互作用的外部调节
所用DNA链序列:
链1:Cholesteryl-CCCTAACCCTAACCCTAACCCATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA-AF 488;
链2:GCAGTTGATCCTTTGGATACCCTGGTTGCAGGTAGAGAAGTrAGGAAACTAT-TAMRA;
链3:BHQ2-GTT AGTGTTAGTGTT AG-Cholesteryl。
(1)我们将链1/链3在94℃下加热5分钟后自然冷却至室温,即可退火形成双链,而后将链1/链3形成的双链在末端胆固醇的疏水作用下插入到T细胞膜表面。而链2中MUC1的适配体可与癌细胞表面的过表达的MUC1结合。之后通过链1、链2和链3之间的杂交实现T细胞/癌细胞组装。
如图2a所示,由链1、链2和链3构建的DNAzyme分子机器通过以下五个步骤应用于T细胞/癌细胞相互作用的外部调节:1.链2识别癌细胞表面MUC-1;2.T细胞与癌细胞的组装;3.酸性pH下的细胞-细胞接近;4.中性pH下T细胞与癌细胞的距离变宽;5.通过Zn2+激活的裂解进行细胞间分解。细胞显微图像证实了与上述五个步骤相对应的T细胞和癌细胞之间细胞-细胞相互作用的动态操纵(图2b)。当细胞混合后,可以立即实现细胞-细胞组装。在细胞-细胞组装后,TAMRA的荧光被BHQ2猝灭(图2b,步骤2)。当我们将溶液pH降至6.0时,TAMRA荧光明显恢复,因为酸性pH诱导链-1折叠,导致BHQ2在链-3的5'端离开(图2b,步骤3)。将pH调回中性后,由于链-1的结构恢复,TAMRA荧光再次熄灭(图2b,步骤4)。接下来,随着Zn2+的加入,底物链2从核糖核苷酸切割位点被切割,导致细胞立即解体(图2b,步骤5)。
(2)在实现细胞间聚集的基础上,通过调节pH值可实现T细胞和癌细胞之间的可逆距离控制(图2c)。如图2d所示,TAMRA在癌细胞膜上的荧光在弱碱性pH值(pH=7.5)下猝灭。当pH值调至酸性(pH=6)时,TAMRA逐渐恢复荧光。在pH值逐渐调整回弱碱性后,荧光强度再次降低。这些结果表明,连接后的T细胞和癌细胞之间的距离可以通过pH控制进行可逆和精确的调节。总之,上述结果证实了基于我们设计的T细胞/癌细胞相互作用的外部调节的可行性。
实施例2.线粒体聚集的内部调节
所用DNA链序列:R8靶向线粒体的多肽
链4:R8-ATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA-TAMRA;
链5:R8-TTGCAGGTAGAGAAGTrAGGAAACTAT-AF 488。
R8:NH2-(Arg)8-CONH2
(1)先进行细胞外线粒体相互作用研究。如图3a所示,从活细胞中提取的线粒体分为两组,其表面分别与链4(用AF 488标记的Zn2+特异性DNAzyme)和链5(用TAMRA标记的底物链)相连。当这两组线粒体混合时,可以形成线粒体聚集。如图3b所示,在共焦显微镜图像下可以清楚地观察到线粒体组装。添加Zn2+离子后,底物链5被裂解,在30分钟内立即断开。TEM图像也证实了上述结果(图3c)。
(2)之后,在活细胞中测试细胞内线粒体相互作用。将链4和链5分别包裹于脂质体中,然后与PANC-1细胞孵育以进行细胞内递送。如图3e所示,当PANC-1细胞用链4(2μL,100μM)处理1小时,然后用链5(2μL,100μM)处理1小时时,观察到广泛的线粒体聚集。在加入Zn2+离子后,在红色和绿色荧光通道下,30分钟后可以清楚地观察到线粒体断开。3D细胞图像(图3f)和TEM图像(图3g)进一步证实了链4和链5处理下细胞内线粒体的聚集。上述结果表明,线粒体聚集程度与细胞内锌浓度有关,因为线粒体聚集更可能发生在缺锌癌细胞中,这有利于精确的癌症治疗。
实施例3.链组一不同组成下的细胞相互作用
(1)在图3a中将链1,链3一端的胆固醇替换成了C6 spacer,在图3b中将链1,链3一端的胆固醇替换成了C12 spacer。
(2)最后按照实施例1中所述步骤,由链1、链2和链3构建的DNAzyme分子机器应用于T细胞/癌细胞相互作用的外部调节。
(3)在图4a中将链1上的imotif修饰成两个,链3上也修饰上两个互补链,在图4b中将链1上的imotif修饰成3个,链3上也修饰上3个互补链。
(4)最后按照实施例1中所述步骤,由链1、链2和链3构建的DNAzyme分子机器应用于T细胞/癌细胞相互作用的外部调节。
实施例4.联合杀死癌细胞的DNAzyme分子机器
(1)最后,在缺锌的PANC-1细胞中进行了联合治疗的试验。如图6a所示,对存活的肿瘤细胞进行Annexin-V-FITC/PI染色,检测以下不同处理条件对肿瘤细胞的杀伤效果:(i)对照组:未经任何处理的PANC-1细胞,(ii)PANC-1细胞与未修饰的t细胞混合,(iii)链-2修饰的PANC-1细胞与链-1/链-3修饰的t细胞混合(该DNAzyme分子机器用于t细胞/癌细胞相互作用的外部控制),(v)PANC-1细胞与脂质体包裹的链-4/链-5混合(该DNAzyme分子机器用于线粒体聚集的内部控制),(v)PANC-1细胞与未修饰的t细胞和脂质体包裹的链-4/链-5混合,(vi)链-2修饰的PANC-1细胞与链-1/链-3修饰的t细胞和脂质体包裹的链-4/链-5细胞(联合治疗)。
(2)随后,Annexin-V-FITC/PI染色检测肿瘤细胞凋亡程度(图6b)。从显微镜图像可以看出,细胞凋亡荧光强度在DNAzyme分子机器内外调控联合作用下最高。图6c-d中对应的荧光强度进一步证实了上述结果。图6e给出了说明该实验原理的模型,揭示了内外联合治疗相互调节的趋势。这些结果表明,所建立的联合治疗具有增强的协同杀伤肿瘤细胞的作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 华东理工大学
<120> 工程化DNAzyme分子机器及其应用
<130> /
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccctaaccct aaccctaacc catagtttct ccgagccggt cgaaacttct ctacctgcaa 60
<210> 2
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcagttgatc ctttggatac cctggttgca ggtagagaag traggaaact at 52
<210> 3
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gttagtgtta gtgttag 17
<210> 4
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
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atagtttctc cgagccggtc gaaacttctc tacctgcaa 39
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttgcaggtag agaagtragg aaactat 27
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccctaaccct aaccctaacc c 21
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<213> 人工序列(Artificial Sequence)
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tccctaaccc taaccctaac ccaa 24
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gttagtgtta gtgttag 17
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<213> 人工序列(Artificial Sequence)
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ggattcgcct ttcgctta 18
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<212> DNA
<213> 人工序列(Artificial Sequence)
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atagtttctc cgagccggtc gaaacttctc tacctgcaa 39
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<213> 人工序列(Artificial Sequence)
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acagacatct cttctccgag ccggctgaaa tagtgagt 38
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<213> 人工序列(Artificial Sequence)
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ttgcaggtag agaagtragg aaactat 27
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<212> DNA
<213> 人工序列(Artificial Sequence)
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actcactatr aggaagagat gtctgt 26
Claims (12)
1.工程化DNAzyme分子机器,其特征在于,包括链组一和链组二中的一组或两组,
链组一:i-motif链修饰的DNAzyme及其底物链,所述链组一修饰在癌细胞与T细胞表面,利用肿瘤细胞的酸性微环境使其修饰的i-motif链收缩,缩短癌细胞与肿瘤细胞间距离;
链组二:线粒体靶向肽修饰的DNAzyme及其底物链,利用细胞内线粒体聚集来诱导细胞凋亡。
2.根据权利要求1所述的工程化DNAzyme分子机器,其特征在于,所述链组一包括三条DNA链,其中链1由长脂链、i-motif链和DNAzyme链组成,链2由识别肿瘤细胞高表达的蛋白适配体和DNAzyme相对应的底物链组成,链3由i-motif的互补链和长脂链组成。
3.根据权利要求2所述的工程化DNAzyme分子机器,其特征在于,所述链1和链2还包括pH不敏感的荧光基团,所述链3还包括猝灭剂。
4.根据权利要求3所述的工程化DNAzyme分子机器,其特征在于,所述荧光基团包括TAMRA、AF 488、CY3、AF 555、AF 532或AF546中的一种。
5.根据权利要求2所述的工程化DNAzyme分子机器,其特征在于,所述长脂链包括胆固醇、C6 Spacer、C12 Spacer或C18 Spacer中的一种。
6.根据权利要求2所述的工程化DNAzyme分子机器,其特征在于,所述识别肿瘤细胞高表达的蛋白适配体包括MUC1适配体、PD-L1适配体或EGFR适配体中的一种。
7.根据权利要求2所述的工程化DNAzyme分子机器,其特征在于,所述链组一的三条DNA链分别为:链1:Cholesteryl-CCCTAACCCTAACCCTAACCCATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA;链2:GCAGTTGATCCTTTGGATACCCTGGTTGCAGGTAGAGAAGTrAGGAAACTAT;
链3:GTT AGTGTTAGTGTT AG-Cholesteryl。
8.根据权利要求1所述的工程化DNAzyme分子机器,其特征在于,所述链组二包括两条DNA链,其中链4由线粒体靶向肽和DNAzyme链组成,链5由线粒体靶向肽和DNAzyme链对应的底物链组成。
9.根据权利要求8所述的工程化DNAzyme分子机器,其特征在于,所述链4和链5还包括荧光基团。
10.根据权利要求8所述的工程化DNAzyme分子机器,其特征在于,所述线粒体靶向肽包括R8:NH2-(Arg)8-CONH2或Fmoc-FFFGKsuccG-COOH中的一种。
11.根据权利要求8所述的工程化DNAzyme分子机器,其特征在于,所述链组二的两条DNA链分别为:
链4:R8-ATAGTTTCTCCGAGCCGGTCGAAACTTCTCTACCTGCAA;
链5:R8-TTGCAGGTAGAGAAGTrAGGAAACTAT。
12.权利要求1—11任一所述工程化DNAzyme分子机器在制备抗肿瘤药物中的应用。
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