CN112778235A - 新型fret供受体对及其应用 - Google Patents
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Abstract
本发明涉及生物分析技术领域，特别涉及新型FRET供受体对及其应用。本发明提供的FRET供受体对呈现出比传统体系(如Cy3/Cy5)更好的FRET效率、光稳定性、耐酸性，并将其作为信号读出体系，适用于酸性条件下靠近诱导的DNA适配体传感分析。
Description
技术领域
本发明涉及生物分析技术领域，尤其涉及新型FRET供受体对及其应用。
背景技术
荧光共振能量转移(FRET)是一种比较成熟的生物分析及细胞成像技术，在DNA分析领域应用最为广泛的FRET体系是花菁染料Cy3/Cy5对。然而，研究表明Cy5荧光受Mg2+影响比较大，但Mg2+在DNA分析中经常被用来稳定DNA纳米脚手架；另一方面，Cy5荧光受酸性pH影响，在酸性环境中工作信号较弱。因此，发展受外界条件影响较小的新型高效FRET体系，在DNA传感分析及细胞生物成像方面具有良好应用前景。
一些噻唑类荧光分子转体如硫磺素(ThT)具有自身荧光低、与某些DNA二级结构特异作用等性质，在DNA传感分析与细胞生物成像方面引起极大关注。由于分子内电子转移作用，这些分子荧光往往表现出较大Stokes位移，在FRET体系中能够有效减小供体荧光对FRET信号的干扰。然而，在前期报道中，很少将其成对用作FRET体系，并普遍用于DNA分析与细胞成像中，主要原因在于：一、能够标记DNA的该类功能化荧光探针较难合成；二，该类探针荧光较弱，只适用于某些特殊DNA结构中，普适性较低。由此可见，开发一种新型高效FRET体系，具有及其重要的现实意义。目前国内外在此方面没有相关解决方法和技术报道。
发明内容
本发明的目的在于提供新型FRET供受体对及其应用，本发明FRET供受体对体系具有更好的FRET效率、光稳定性和耐酸性。
本发明提供的FRET供受体对，包括供体化合物和受体化合物；
所述受体化合物具有式I所示结构，供体化合物具有式II所示结构，
其中，R1、R1’独立的选自氢、C1～C6的烷基，R2、R2’独立的选自选自氢、苯基、烷基取代的苯基、卤素取代的苯基，R3、R3’独立的选自C1～C6的烷基、烷基取代的磺酸基。
一些实施方案中，R1、R1’独立的选自氢、甲基。
一些实施方案中，R2，R2’独立的选自氢、苯基；R2，R2’为苯基时，R2，R2’分别与其连接的苯环形成稠环结构。
一些实施方案中，R3，R3’独立的选自甲基、乙基、丙磺酸基。
一些优选实施例中，R1，R1’为氢；R2、R2’为苯基，R2、R2’分别与其连接的苯环形成稠环结构；R3、R3’分别为丙磺酸基团与甲基，具体地，受体化合物结构如式I-1所示，将其命名为WL2.5；供体化合物的如式II-2所示，将其命名为CNT:
一些优选实施方案中，R1，R1’独立的选自氢、甲基，R2、R2’为氢或苯基；R2、R2’为苯基时，R2、R2’分别与其连接的苯环形成稠环结构；R3、R3’选自丙磺酸基、甲基、乙基。，具体地，受体化合物结构如式I-2～6所示，供体化合物的如式II-2～6所示:
本发明FRET供受体化合物的制备过程中，原料和合成方法很关键。对萘并噻唑上的N原子进行带羧基的取代反应为本领域一般的合成路径，然而此步骤产率低，分离提纯困难。本发明改进了羧基化路线，采用带氰基的原料(氰乙基甲基氨基苯甲醛)来合成荧光分子转体，再将氰基水解成羧基，经过一系列反应生成DNA荧光染料包括发绿光的荧光供体化合物和发红光的荧光受体化合物，如荧光供体化合物CNT(如式I-1所示结构)和荧光受体化合物WL2.5(如式II-1所示结构)。
具体的，本发明还提供了FRET供受体对的制备方法，所述受体化合物的制备方法为：
将式I-a所示化合物和丙磺酸内脂混合，反应得到式I-b所示化合物；在乙醇回流条件下，式I-b所示化合物和羧乙基甲基氨基苯甲醛反应，得式I所示化合物；
所述羧乙基甲基氨基苯甲醛由氰乙基甲基氨基苯甲醛水解得到。
所述供体化合物的制备方法为：
将具有式II-a结构的化合物和碘化烷在有机溶剂存在的条件下，加热反应得到式II-b所示化合物；II-b所示化合物和氢氧化钾在乙二醇回流条件下反应获得II-c所示化合物；II-c所示化合物和羧乙基甲基氨基苯甲醛在还原剂存在的条件下经乙醇回流反应，得式II所示化合物；
所述羧乙基甲基氨基苯甲醛由氰乙基甲基氨基苯甲醛水解得到。
本发明还提供了所述FRET供受体对在制备荧光探针以及生物传感器中的应用。
本发明还提供了所述FRET供受体对在核酸分析检测、细胞成像分析中的应用。
基于本发明提供的FRET供受体，本发明还提供了标记有所述FRET供受体对的荧光探针。进一步，本发明还提供了利用该荧光探针制成的生物传感器。具体的，所述生物传感器包括ATP适配体传感器。
与现有技术相比，本发明提供的FRET供受体具有如下优势：
(1)本发明FRET体系即CNT/WL2.5对，标记在双链DNA末端的FRET效率高达86.7％，作为对照，相同条件下传统Cy3/Cy5体系的FRET效率为58.0％；另外，在光稳定性和耐酸性等方面，CNT/WL2.5体系性能均优于Cy3/Cy5对。
(2)该FRET体系的Stokes位移在200nm左右，而传统的Cy3/Cy5体系在120nm左右，较大的Stokes位移不仅能够有效避免供体荧光对FRET信号的干扰，在用于细胞及活体成像时，也能够极大地降低背景荧光。
附图说明
图1示本发明FRET供受体对合成的荧光探针的光谱性质及FRET性能；
图2示本发明FRET体系在DNA适配体传感分析中的应用。
具体实施方式
本发明公开了FRET供受体对及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
对所公开的实施例的说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
下面结合实施例，进一步阐述本发明：
实施例1本发明FRET供受体对的制备
如式I-1所示的受体化合物WL2.5，如式II-1所示的供体化合物CNT，其合成路线如下：
(1)氰乙基甲基氨基苯甲醛在含过氧化氢的NaOH水溶液中，氰基水解成羧基，获得羧乙基甲基氨基苯甲醛；
(2)从2-甲基萘并噻唑出发，按照以上合成路线进行反应，得到FRET体系中的荧光供体、受体，即CNT和WL2.5。
其中，WL2.5合成过程：取2.14克2-甲基萘并噻唑加入到2.75mL丙磺酸内脂中，在140度油浴中搅拌6h，将得到产物用甲醇洗涤后，取1.605克产物于20mL乙醇中，加入1.03克羧乙基甲基氨基苯甲醛，搅拌回流10h，用冷乙醚沉淀，得到暗紫色产物，产率60％。
CNT合成过程：取2.14克2-甲基萘并噻唑于6mL DMF中，加入1.86mL碘甲烷，在80度油浴中搅拌过夜，产物用冷乙醚沉淀，然后，取1.7g产物加入到10mL乙二醇中，并加入30mL50％KOH，搅拌回流24h后，并在室温下继续搅拌24h，产物用HCl中和后，以二氯甲烷萃取，最后，取0.376克产物加入到25mL乙醇中，并加入0.41克羧乙基甲基氨基苯甲醛，搅拌回流6h，用冷乙醚沉淀后，用高效液相色谱分离，得到黄色产物，产率25％。
实施例2本发明FRET供受体对的制备
式I-2～3所示的供体化合物ThT-C，如式II-2～3所示的受体化合物WL-2，其合成路线如下：
(1)氰乙基甲基氨基苯甲醛在含过氧化氢的NaOH水溶液中，氰基水解成羧基，得羧乙基甲基氨基苯甲醛；
(2)从2-甲基萘并噻唑出发，按照以上合成路线进行反应，分别得到式I-2～3所示的荧光供体化合物和如式II-2～3所示的受体化合物。
实施例3本发明FRET供受体对的光谱性质及FRET性能
本发明实施例1提供的FRET供受体对CNT和WL2.5的吸收与荧光光谱，呈现近200nm的Stokes位移，另外，CNT的荧光发射光谱与WL2.5的吸收光谱重合较好，如图1a所示。CNT与WL2.5的FRET效率很好，远超过传统的Cy3/Cy5体系，如图1b所示。另外，CNT/WL2.5体系的光稳定性也优于Cy3/Cy5对，如图1c所示。
实施例4本发明FRET供受体对在ATP分析检测中的应用
在DNA三臂、四臂二聚结构上分别连接ATP适配体的两部分，将CNT、WL2.5分别标记到ATP适配体末端，该体系在i-motif折叠作用下，通过二聚组装，使ATP适配体的两部分靠近，从而与ATP分子作用，与ATP作用后，CNT和WL2.5靠近，产生较强FRET信号，从而实现ATP分析检测，参见图2。
若将DNA三臂、四臂结构嫁接到四面体顶点上，该体系可进入细胞体内，对溶酶体ATP进行成像分析。
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来说，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。
Claims (10)
1.FRET供受体对，包括供体化合物和受体化合物；
所述受体化合物具有式I所示结构，供体化合物具有式II所示结构，
其中，R1、R1’独立的选自氢、C1～C6的烷基，R2、R2’独立的选自氢、苯基、烷基取代的苯基、卤素取代的苯基，R3、R3’独立的选自C1～C6的烷基、烷基取代的磺酸基。
2.根据权利要求1所述的FRET供受体对，其特征在于，R1、R1’独立的选自氢、甲基。
3.根据权利要求1所述的FRET供受体对，其特征在于，R2，R2’独立的选自氢、苯基；R2，R2’为苯基时，R2，R2’分别与其连接的苯环形成稠环结构。
4.根据权利要求1所述的FRET供受体对，其特征在于，R3，R3’独立的选自甲基、乙基、丙磺酸基。
5.根据权利要求1所述的FRET供受体对，其特征在于，所述受体化合物具有式I-1～I-6所示的结构，所述供体化合物具有如式II-1～II-6所的结构；
6.权利要求1～5任一项所述的FRET供受体对的制备方法，其特征在于，所述受体化合物的制备方法为：
将式I-a所示化合物和丙磺酸内脂混合，反应得到式I-b所示化合物；在乙醇回流条件下，式I-b所示化合物和羧乙基甲基氨基苯甲醛反应，得式I所示化合物；
羧乙基甲基氨基苯甲醛由氰乙基甲基氨基苯甲醛水解得到。
7.权利要求1～5任一项所述的FRET供受体对的制备方法，其特征在于，所述供体化合物的制备方法为：
将具有式II-a结构的化合物和碘化烷在有机溶剂存在的条件下，加热反应得到式II-b所示化合物；II-b所示化合物和氢氧化钾在乙二醇回流条件下反应获得II-c所示化合物；II-c所示化合物和羧乙基甲基氨基苯甲醛在还原剂存在的条件下经乙醇回流反应，得式II所示化合物；
所述羧乙基甲基氨基苯甲醛由氰乙基甲基氨基苯甲醛水解得到。
8.权利要求1～5任一项所述的FRET供受体对或权利要求6或7所述制备方法制得的FRET供受体对在制备荧光探针以及核酸传感分析、细胞成像分析中的应用。
9.荧光探针，其特征在于，所述荧光探针标记有权利要求1～5任一项所述的FRET供受体对。
10.包含权利要求9所述荧光探针的生物传感器。
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