CN110546276A - 用于分子传感器的酶电路 - Google Patents
用于分子传感器的酶电路 Download PDFInfo
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- CN110546276A CN110546276A CN201880026531.4A CN201880026531A CN110546276A CN 110546276 A CN110546276 A CN 110546276A CN 201880026531 A CN201880026531 A CN 201880026531A CN 110546276 A CN110546276 A CN 110546276A
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Abstract
在多个实施例中,公开了一种分子电路。该电路包括负电极、与负电极间隔开的正电极、以及导电地附接至正电极和负电极两者从而形成具有通过酶的导电通路的电路的酶分子。在多个示例中,该酶是聚合酶。该电路还可包括用于将酶接线至电极的分子臂。在多个实施例中,该电路用作传感器,其中在底物与酶相互作用时测量电信号,例如电路中的电压、电流、阻抗、电导率或电阻的变化。
Description
对相关申请的引用
本申请要求于2017年4月25日提交的名称为“用于分子传感器的酶电路”的美国临时专利申请62/489,881的优先权和权益,该临时专利申请的公开内容通过完整引用结合在此。
技术领域
本公开总体地涉及分子传感器,更具体地说,涉及一种在其中由酶闭合两个电极之间的电路的分子传感器。
背景技术
在二十世纪七十年代,艾维拉姆和拉特纳带来了分子电子学的广阔领域。分子电子学通过使用单个分子作为电路元件实现了电路的终极缩小。根据分子的性质,包含单分子元件的分子电路可用作开关、整流器、致动器和传感器。特别令人感兴趣的是这种电路作为传感器的应用,其中分子相互作用为单分子传感提供了基础。尤其是,信息性的电流变化可包括电流的增大或减小、脉冲、或其他时间变化。
虽然在分子电子学领域取得了不少成就,但仍需要可用作分子传感器的新分子电路。尤其是,仍需要一种改良的单分子***,这种***能够产生具有更大信噪比的分子信息,从而使真正表示分子相互作用的信号与非信息性噪声区分开来。
发明内容
在多个实施例中,公开了一种基于单分子酶的电路,其中单个酶分子直接连接至正电极和负电极以形成电路。这些电路能够产生高度信息性的酶活性信号。这些改良信号具有更高的信噪比水平,因此信号与噪声之间的区别更大,并且这些改良信号包括携带关于酶与目标底物之间的结合的详细信息的特征。
在多个实施例中,一种分子传感器包括如本文所述的基于酶的分子电路(导电通路)。这种具有聚合酶的传感器可用于从由聚合酶处理的DNA模板感测序列信息。
在本公开的多个实施例中,公开了一种分子电路。该电路包括:正电极;与正电极隔开的负电极;以及连接至正电极和负电极从而在正电极与负电极之间形成导电通路的酶。
在多个方面中,所述电路的酶可包括连接至正电极的第一接线点和连接至负电极的第二接线点。
在多个方面中,所述电路还可包括具有第一端和第二端的至少一个臂分子,所述第一端结合至酶,所述第二端结合至所述电极中的至少一个,其中所述至少一个臂分子作为酶与所述电极中的至少一个之间的电线。
在多个方面中,所述至少一个臂分子可选自由双链寡核苷酸、肽核酸双链体、肽核酸-DNA杂化双链体、蛋白质α螺旋、类似石墨烯的纳米带、天然聚合物、合成聚合物和抗体Fab结构域组成的组。
在多个方面中,所述电极中的至少一个连接至酶的内部结构元件。
在多个方面中,所述内部结构元件可选自由α螺旋、β褶板和串联的多个这种元件组成的组。
在多个方面中,所述电极中的至少一个可在酶的能够发生构象变化的位置连接至酶。
在多个方面中,至少一个臂分子可包括具有取决于张拉、扭曲或扭转的电导率的分子。
在多个方面中,所述酶可包括聚合酶。
在多个方面中,所述聚合酶包括大肠杆菌Pol I Klenow片段。
在多个方面中,所述聚合酶包括逆转录酶。
在多个方面中,所述聚合酶包括经过基因修饰的逆转录酶。
在多个方面中,一种分子传感器包括电路,该电路进一步包括:正电极;与正电极隔开的负电极;以及聚合酶,该聚合酶包括连接至正电极和负电极从而在正电极与负电极之间形成导电通路的大肠杆菌Pol I Klenow片段,其中所述正电极和所述负电极均在位置514和547的氨基酸之间延伸的聚合酶的主α螺旋内的连接点处连接至聚合酶。
在多个方面中,一种分子传感器包括电路,该电路进一步包括:正电极;与正电极隔开的负电极;以及连接至正电极和负电极从而在正电极和负电极之间形成导电通路的聚合酶,其中所述传感器可用于从由聚合酶处理的DNA模板感测序列信息。
在多个方面中,一种分子传感器包括电路,该电路进一步包括:正电极;与正电极隔开的负电极;以及连接至正电极和负电极从而在正电极与负电极之间形成导电通路的聚合酶,其中所述正电极和所述负电极均在聚合酶的拇指和食指结构域的连接点连接至聚合酶,并且其中这些点随着聚合酶处理DNA模板而发生超过1纳米的相对运动。
在多个方面中,所述传感器中的聚合酶被改造为具有延长结构域,该延长结构域随着聚合酶处理DNA模板而产生更大范围的相对运动。
在多个方面中,所述传感器中的聚合酶被改造为具有附加的电荷基团,这些附加的电荷基团在酶处理DNA模板时可变地影响内部导电通路。
在多个方面中,所述电路中的聚合酶是大肠杆菌、Pol I、Bst、Taq、Phi29或T7DNA聚合酶的基因修饰形式,或者是经过基因修饰的逆转录酶。
在多个方面中,一种分子电路包括:正电极;与正电极隔开的负电极;以及连接至正电极和负电极从而在正电极与负电极之间形成导电通路的酶,其中所述正电极和所述负电极均在所述酶中的连接点处与连接至酶,所述连接点包括天然半胱氨酸、基因工程半胱氨酸、具有接合残基的基因工程氨基酸、或包括具有接合配偶的肽的基因工程肽结构域中的至少一种。
在多个方面中,所述电路还包括栅电极。
在多个实施例中,公开了一种对DNA分子进行测序的方法。所述方法包括:提供电路,该电路进一步包括正电极、与正电极隔开的负电极、以及与正电极和负电极连接从而在正电极与负电极之间形成导电通路的聚合酶;引发通过所述电路的电压或电流中的至少一种;使所述电路暴露于含有引物单链DNA和/或dNTP的溶液;以及在聚合酶接合并延长模板时,测量通过所述电路的电信号,其中对所述电信号进行处理以识别提供关于由聚合酶处理的DNA分子的基础序列信息的特征。
在多个实施例中,公开了一种分子检测方法。该方法包括,提供电路,该电路进一步包括正电极、与正电极隔开的负电极、以及连接至正电极和负电极从而在正负电极与栅电极之间形成导电通路的聚合酶;引发通过所述电路的电压或电流中的至少一种;使电路暴露于以下条件中的至少一种:离子强度降低的缓冲液、包括经过修饰的dNTP的缓冲液、包括变化的二价阳离子浓度的缓冲液、施加在主电极上的特定电压、栅电极电压、或施加在主电极或栅电极上的电压谱或扫描;以及测量所述电路中的电气变化。
附图说明
在说明书的总结部分中特别指出并明确要求保护本公开的主题。但是,通过结合附图参阅详细说明和权利要求,能够最佳地获得对本公开的更全面的理解,在附图中:
图1示出了分子电子电路的一般概念;
图2示出了酶与分子电子电路接合(例如与其目标物接合)作为酶活性传感器的一般概念;
图3示出了多个实施例的直接接线至电流路径的酶;
图4示出了多个实施例的直接接线至电流路径的酶,其中所述连接是连接至酶中的内部α螺旋结构;
图5示出了多个实施例的直接接线至电流路径的酶,其所述连接是连接至酶中串联的两个或更多内部α螺旋结构;
图6示出了多个实施例的直接接线至电流路径的酶,其中所述连接是连接至酶中的内部β褶板结构;
图7示出了直接接线至电流路径的酶,其中所述连接是连接至酶中的构象变化点,以在酶活跃期间在电路中引起张力变化;
图8示出了直接接线至电流路径的酶,其中形成了附加连接,以稳定酶的位置;
图9示出了多个实施例的直接接线至电路的电流路径的酶的示意图,其中所述酶直接耦合至电极,而不使用臂分子;
图10示出了多个实施例的通过两个接触点直接接线至电路的酶的示意图,并且所述酶还具有与分子线的单点接合,从而利用一对电极来测量联合传导;
图11示出了多个实施例的通过两个接触点直接接线至电路的酶的示意图,并且所述酶还具有与分子线的单点接合,从而利用两对电极来独立地测量这两个传导模式;
图12示出了大肠杆菌Pol I Klenow片段聚合酶的蛋白质结构图,其中示出了α螺旋、β褶板和连接环结构的存在;
图13示出了多个实施例的直接接线至电路的电流路径的聚合酶的示意图,其中特定的α螺旋用于接触点,并且分子臂提供至电极的耦合;
图14示出了多个实施例的直接接线至电路的电流路径的聚合酶的示意图,其中特定的α螺旋用于接触点,并且该聚合酶直接耦合至电极,而不使用臂分子;
图15示出了多个实施例的直接接线至电路的电流路径的聚合酶的示意图,其中当食指和拇指结构域改变相对构象时,臂接线至发生相对运动的点;和
图16示出了直接接线至电路的电流路径的聚合酶的示意图,并且其中连接有附加的连接臂,以实现稳定化和固定的空间取向。
具体实施方式
在此参照附图对示例性实施例进行详细说明,这些附图以示意性方式示出了示例性实施例以及其最佳模式。虽然对这些示例性实施例的说明足够详细,能使本领域技术人员实践在此详述的发明,但是应理解,在不脱离本发明的精神和范围的前提下,还可实现其他实施例,并且可做出逻辑、化学和机械变化。因此,在此给出的详细说明仅是出于示例性目的,而不是限制性的。例如,除非另有说明,否则在任何方法或过程说明中叙述的步骤可按任何顺序执行,并且不必受限于所给出的顺序。此外,对单数的任何引用包括多个实施例,并且对不止一个部件或步骤的任何引用可包括单个实施例或步骤。而且,对附接、固定、连接等的任何引用可包括永久的、可移除的、临时的、部分的、完全的和/或任何其他可能的附接方案。另外,对无接触(或类似短语)的任何引用也可包括减少接触或最少接触。
在本公开的多个实施例中,公开了一种分子电路。该分子电路包括:正电极;与正电极隔开的负电极;以及连接至正电极和负电极从而在正电极与负电极之间形成导电通路的酶。在多个示例中,所述酶包括连接至正电极的第一接线点和连接至负电极的第二接线点。
定义和解释
在本文所用的术语“酶”指通过与不同底物分子接合而起作用以转化另一个分子的分子。这种转化可包括化学修饰或构象修饰。常见的生物酶类别有聚合酶、连接酶、核酸酶、激酶、转移酶、以及这些分子的基因修饰形式。在此所述的聚合酶包括能够直接作用于RNA模板的逆转录酶和任何经过遗传修饰的逆转录酶。酶是最常见的蛋白质,但可由多个氨基酸链组成,也可与其他类型的分子复合,例如在核糖体酶的情况下,可与RNA复合。
在本文所用的术语酶的“底物”指酶在进行转化过程中与之特异性接合的任何分子。例如,在DNA聚合酶的特定情况下,所述底物由模板DNA和dNTP组成。除了酶的底物,酶还可与各种调节其功能或动力特性的辅助因子复合。例如,在DNA聚合酶的情况下,二价阳离子(例如Mg++)通常是必不可少的辅助因子,但不能视为底物。
在本文所用的术语“dNTP”指参与基于聚合酶的DNA合成或可用于这种DNA合成的任何脱氧核苷酸三磷酸,包括此类分子的原生形式和修饰形式。
在本文所用的术语“酶缓冲液”指酶在其中有活性并起作用并且通常包含保持酶活性所需的底物和辅助因子的溶液。这种酶缓冲液通常可包括单独的或各种组合形式的盐、去污剂和表面活性剂、以及特定的辅助因子(例如用于聚合酶的镁或其他二价阳离子)和底物(例如用于聚合酶的DNA和dNTP)。这种缓冲液在此可具有从标准形式修改的组成,以增***露于该缓冲液的传感器中的信号特性等。
在本文所用的术语“电极”指可作为载荷子的高效源或汇的任何结构。其中最常见的是金属或半导体结构,例如在电子电路中使用的结构。在此所述的一对间隔开的电极可包括源电极-漏电极对。在本公开的多个实施例中,基于结合探针的分子电路可进一步包括栅电极。当存在时,栅电极用于施加电压而不是转移载荷子。因此,它支持载荷子的积累以产生局部电场,但不旨在传递电流。栅电极通过某种形式的绝缘层或材料与电路的主导电通路电隔离。
在本文所用的术语“接合”是指将一个分子物理附接至另一个分子或者表面或粒子的多种手段中的任何一种。此类方法通常涉及形成共价或非共价化学键,但也可能依靠蛋白质之间的相互作用、蛋白质与金属之间的相互作用、或者通过分子间力(范德华力)的作用实现的化学或物理吸附。接合化学领域的技术人员知道许多此类方法。与在此所述的优选实施例相关的常见接合方法包括硫醇-金属键、马来酰亚胺-半胱氨酸键,材料结合肽(例如金结合肽)和点击化学。
在本文所使用的术语“引发”在电气参数的背景下比“施加”电气值的概念更宽泛。例如,可在电路中引发电流。电流的这种引发可能是向电路施加电压的结果,但是,也可能是除了施加电压之外的其他动作导致的。此外,可在电路中引发电压。电压的这种引发可能是向电路施加电流的结果,但是,也可能是除了施加电流之外的其他动作导致的。在其他示例中,作为向整个电路施加电压或电流的结果,可在电路的一部分中引发电压或电流。在一个非限制性示例中,可通过施加到电路的栅电极的电压来控制在本公开的电路中引发的电子从负电极向正电极的流动。
在本公开的多个实施例中,分子传感器包括连接至正电极和负电极以构成电路的酶。酶与各种底物的相互作用可作为整个电路中测量的电流或其他电气参数的变化检测。本发明的分子传感器与分子电子电路的一般概念的不同之处在于,酶直接“接线”至正电极和负电极,而不是键合至跨越电极之间的间隙的分子桥分子,以形成一个完整的电路。
在本公开的各个方面中,在基于酶的分子电路中引发电压或电流中的至少一种。当目标物与酶相互作用时,可感测到电路中的电气变化。这些电气变化或信息性电信号可包括电流、电压、阻抗、电导率、电阻、电容等。在一些示例中,在电路中引发电压,然后在底物与酶相互作用时测量通过电路的电流的变化。在其他示例中,在电路中引发电流,然后在底物与酶相互作用时测量通过电路中的电压的变化。在其他示例中,测量阻抗、电导率或电阻。在电路还包括栅电极(例如位于正负电极之间的间隙下方)的示例中,可向栅电极施加电压或电流中的至少一种,并且当底物与酶相互作用时可测量电路中的电压、电流、阻抗、电导率、电阻或其他电气变化。
图1示出了分子电子电路的一般概念,该分子电子电路具有附接至并桥接电极15之间的间隙12的桥分子10、以及将分子结合至电极的某种类型的接合基团20或其他机构(描绘为带阴影的小方块)。图1还示出了电流(i)可通过该分子,并可测量电流(i)与时间(t)的关系,如插图25所示。
图2示出了一种分子电子传感器,其中酶30与跨电极32的分子桥元件31接合,其中当酶暴露于适当的缓冲溶液时,监测电流提供了一种与其目标底物35接合并对其目标底物35进行处理的酶传感器。在这种传感器***中,由目标底物与酶接合引起的局部电荷微扰对通过主电桥元件进行的电荷传送产生微扰,因此被记录为电导率或电流随时间的变化,如图2中电流(i)与时间(t)的电流插图38中的台阶状变化所示。
与图1和图2中所示的一般分子电路概念相反,在本公开的多个实施例中,分子传感器包括直接接线至电路路径的单个酶分子,从而经过分子电路的所有电流必须流过酶。因此,所述酶是电路中的基本导电路径,就像电路板上的电子元件一样。本发明的概念总体上在图3中示出,其中示出了连接在分子臂40之间的酶42。通过迫使电路中的所有电流流过酶,载流子被迫在更靠近酶与目标底物45之间的电化学相互作用的精确位置通过,从而使这种相互作用对载流子产生更大的影响,并且又使总电流对这些相互作用的细节更加敏感。这由图3中的电流与时间(i与t)的曲线插图50示意性地示出,其中,所示出的电流阶越比由图2的配置产生的电流阶越大得多,并且还包括在如图2所示的电流与时间关系图中不存在的附加特征。较高的电流阶跃能改善信号传导。本文中的相关方法和优选实施例促进了基于酶的分子传感器的信号传导的改善。此外,作为基本导电路径的酶的构型与图2的通用构型有本质的不同,在图2中,有许多不通过酶的导电路径,并且可能没有载流子实际穿过酶,而且没有提供任何手段来引导载荷子通过酶中的关键活性位点附近。
在多个实施例中,所述酶可在两个或更多点处耦合至正电极和负电极,以确保穿过分子结构的载荷子进入酶并从酶出来。
如图3的实施例中所示,两个分子臂与酶接合以提供物理锚点以及电流通过酶的进出路径。这样的臂可包括在酶与电极之间提供导电连接或半导电连接的任何方便的分子。此外,分子臂可提供跨越长度延伸量,以帮助跨越比酶的三维结构宽的较大电极间隙55。这样的臂还可提供在可能与电极发生不利或破坏性的相互作用的位置(例如变性吸附至电极)使酶远离并不与电极65接触的优点。这样的臂还可使与电极的耦合的相容性更好或更高效,例如经由在酶上不容易发现或获得的化学基团耦合至电极。例如,在一个特定实施例中,电极包括金,并且分子臂包括硫醇基,从而所述臂通过众所周知的硫醇-金结合耦合至金电极。因此,分子臂实现了结合,而酶可能没有这种可用的巯基。或者,在另一个实施例中,所述臂可具有点击化学结合基团,用于耦合至通过同源结合配偶衍生的点击化学电极。
在多个实施例中,分子臂包括与酶的某种形式的接合60,以及它们与电极的接合或耦合。许多接合化学方法可用于此目的。在一个非限制性示例中,这种接合包括化学交联,该化学交联可优先将臂上的适当化学基团耦合至酶上的氨基酸残基。在多个实施例中,所述臂上的马来酰亚胺基团耦合至酶上的表面半胱氨酸。在其他方面中,可产生和使用酶的遗传修饰形式,例如包括被改造为提供特定接合位点的氨基酸结构的特定氨基酸或蛋白质结构域的酶。例如,在酶的特定位点改造的半胱氨酸氨基酸提供了呈现马来酰亚胺基团的臂的附接点。两个这样的半胱氨酸位点接合至两个马来酰亚胺衍生的臂,从而产生如图3所示的构型。在这种情况下,可从氨基酸序列“改造”出一种或多种提供竞争性臂结合位点的天然半胱氨酸。如果不能去除所有这些位点,那么可使用合成化学中的多种纯化方法从不需要的构型中分离出所需的酶臂接合物。在其他变化形式中,使用遗传方法改造为包括唯一地与臂上的同源基团接合的残基的酶氨基酸序列。这种变化包括使用非标准氨基酸的情况,例如通过蛋白质表达***修饰为呈现点击化学基团的氨基酸,该蛋白质表达***使用经过修饰的遗传密码和经过修饰的转移RNA将非原生氨基酸在表达的酶蛋白中置于特定序列位点。
在其他实施例中,将与所述臂上的同源基团特异性结合的肽结构域改造为蛋白酶序列。在一个这样的实施例中,将作为抗体的抗原的肽改造为酶,并且在所述臂上使用该抗体的Fab结合结构域。一个这样的实施例是使用FLAG肽基序DYKDD和任何适当的ANTI-FLAGFab结构域。通过将肽抗原改造为酶上的所需结合位点,可按类似的方式使用任何其他肽抗原及其同源Fab结构域将所述臂接合至改造的酶蛋白中的特定位点。通过将SPY-TAG结构域或SPY-CATCHER结构域改造为酶蛋白,并将同源结构域置于所述臂中,其他此类肽结构域可利用SPY-TAG/SPY-CATCHER蛋白质间结合***。当将这样的肽结合结构域改造为酶时,另一个实施例是添加作为目标肽的侧翼的短接头肽序列,以增强用于结合的结构域的可用性。如蛋白质工程领域的技术人员所知的,此类短接头可包括短甘氨酸和富含丝氨酸的接头,包括但不限于接头氨基酸序列G、GS、GSG、GGSG等。
在多个示例中,所述臂分子包括为载流子进出酶提供传导作用的任何分子。在一些实施例中,这种臂包括分子电子学领域中已知的多种形式的分子线,这种分子线通过适当的接合和结合基团功能化,以接线至电极和酶。在多个方面中,这种臂可包括单链DNA、双链DNA、肽、肽α螺旋、抗体、抗体的Fab结构域、碳纳米管、石墨烯纳米带、天然聚合物、合成聚合物、具有用于电子离域的p轨道的其他有机分子、或者金属或半导体纳米棒或纳米颗粒。在另一些实施例中,所述臂可包括在一端具有承载巯基的基团并在另一端具有耦合至酶的马来酰亚胺的双链DNA、或者在一端具有半胱氨酸或金结合肽并在另一端具有耦合至酶的马来酰亚胺的肽α螺旋、或者在一端具有承载巯基的基团并在另一端具有耦合至酶的承载马来酰亚胺的基团的石墨烯纳米带。在一些实施例中,所述用于将酶耦合至两个电极的两个臂分子是相同的分子,而在其他实施例中,这两个臂分子是不同的分子。在一些示例中,可存在“正电极”臂和“负电极”臂,以将酶定向结合至图3中的对应“正”电极和“负”电极。
在多个实施例中,臂结合点直接连接至酶内的特定蛋白质结构元件。在图4中示出了一个非限制性的示例,其中臂75被示出直接接线至酶72中的α螺旋结构70。这种结构元件提供了通过酶的优先导电路径。直接接线至酶内的自然导电路径可将电流导引至更靠近酶内的所关注的活性区域(例如底物结合袋),从而可提供进一步增强的电流信号或者载有更多关于酶-底物相互作用的信息的电流信号。例如,图4中示出了一个实施例,其中所述臂直接接线至横跨酶表面上或附近的两个点之间的α螺旋。在图5中示出了另一个示例,其中所述臂80直接接线至酶内部的串联的两个α螺旋(第一α螺旋85和第二α螺旋87),并且单个连接环90将这两个α螺旋分开。在图6中示出了另一个实施例,其中所述臂95直接接线至酶102内部的β褶板100上的两个点98。
通常,蛋白酶具有包含众所周知的二级结构元件(例如α螺旋和β褶板)的三维结构。这些二级结构元件主要是氢键连接结构,它们可提供穿过酶的主体的分立导电路径,使得载流子(例如电子)能高效地沿着此类结构或限定此类结构的氢键跳跃,与跳出或隧穿此类结构的情况相比,这种跳跃的阻力较小。这些结构提供了可导引载荷子的优先导电路径,并且,通过选择此类结构,电荷被迫在靠近酶的活性区域的位置通过,并且能改善基于电流的活性感测。通过使所述臂直接连接至这种结构或在这种结构的端部的少量氨基酸内,能使沿着这些期望路径流动的电流最大化,因而使来自沿着这些路径流动的电流的期望信号最大化。通过这种方式,能最大限度地减少在酶内的其他位置通过的电流,因而还能最大限度地减少因对这些信息性较弱的区域进行探测而引起的噪声。
在多个示例中,所述接线可以是以“串联”形式出现在酶中的结构,例如,如图5所示的两个串联的α螺旋、或与α螺旋串联的β褶板、或三个连续的α螺旋。通常,串联的每个连续元件在酶的主氨基酸序列中表现为通过数个氨基酸与前一个元件隔开,例如0、1、2或最多大约10个氨基酸,这些氨基酸通常在二级结构中形成连接环。串联元件的接线也可通过接线至以下结构实现:该结构在酶的主氨基酸序列中不连续但在空间上连续且导电接触,并因氢键、盐桥、二硫桥或其他类型的分子相互作用而形成优选导电路径的结构桥,所述分子相互作用形成二级、三级或四级蛋白质结构,并且可提供从一个结构元件(β褶板、α螺旋)至另一个结构元件的明确限定且有利的导电路径。当检查所涉及的蛋白质的三维结构时,从晶体结构能够观察到,尤其是在检查通过X-射线或NMR晶体照相术获得的蛋白质结构能够观察到,无论这些用于接线的结构元件是隔离的还是串联的,它们都最为明显。这种有用的结构信息形式由图12中所示的聚合酶结构示出。
在其他实施例中,所述臂接线至酶上的点,所述点在酶起作用期间发生构象变化或相对运动,如图7所示。在这种情况下,臂105接线至如图所示在酶活跃期间具有相对运动的两个点110,如插图108所示。这种构造能通过多种方式增强信号传导。首先,这种运动能改变所述臂的张力,并且,众所周知的是,分子中的张力变化能改变其电导率,因此所述运动可通过张力转换为臂的电导率变化,因而在电流信号中显现出来。通过这种方式,电流可包含关于酶中的构象变化的信息。其次,类似地,这种构造在改变构象时能在酶中引起张力,从而改变酶的电导率。由于酶是基本电流路径,因此构象变化会转换为电流变化,从而在传感电流中表现构象信息。这种构造还可通过改变酶的构象变化来增强信号传导,这在某些情况下可能导致原生酶或经过改造而专门受益于这种构象敏感接线的酶的增强信号。在一个实施例中,对酶进行改造以使其具有发生更大构象变化或相对运动的延长结构域(例如,如通过延长图7所示的剪刀形酶的两个末端的长度所示),以增大运动范围,从而增大所述臂和所述酶中的张力变化范围。
在其他方面中,酶的构象变化(例如当在酶与底物之间发生诱导结合时)被转化为至少一个臂的扭曲、扭转或旋转,并且该扭曲、扭转或旋转改变所述臂的电导率。一个这样的示例是包括有机聚合物的臂,该有机聚合物进一步包括多环芳环,例如聚噻吩或聚亚苯基,其中在所述臂响应于酶的构象变化而扭曲、扭转或旋转时,先前排成一线的p轨道通过CC键旋转而旋转错位。在所述臂扭曲、扭转或旋转时,电子阻止有机聚合物中的离域。在一些实施例中,这种受阻流动可仅作用于一部分载荷子上,这取决于载荷子的极化或其他量子态等,例如电子载荷子的自旋极化、或载荷子的动量或能态。
在图8中示出了另一个示例,其中分子传感器电路包括不止两个臂,例如3个臂(第一臂115、第二臂116和第三臂117)。使用额外的酶接线点和相关的臂的益处包括:增加了酶中的其他所需导电路径,并提高了酶中的总体传导效果。这种额外的臂还能提供稳定性,强制空间曲向(例如强制活性位点的取向),或降低物理自由度或构象熵,这可通过减少具有更多可用构象的***的传导变化来改善感测。这种额外的臂可以是导电的,但如果它们主要是为了提供稳定性,控制取向或减少空间自由度,那么它们也可以是绝缘的。这种额外的臂可连接至电极,或者连接至结构的其他部分,例如连接至支撑电极的底物。在包括不止两个电极的***中(包括具有栅电极(例如埋栅电极)的***的情况),这样的臂可连接至附加的电极。与栅电极的连接可指与栅的导电部分的连接,也可指与将实际的导电栅与电路分开的绝缘层的连接,或者,在埋栅的情况下,指埋栅上方的表面层,例如与图16中所示的表面的连接。
如图9所示,所述酶可直接连接至电极120,作为基本导电路径,而无需使用臂分子。在这种情况下,酶上的基团直接耦合至电极。或者,在另一个实施例中,一个接线连接包括直接耦合至酶,而另一个接线连接经由臂分子实现。这种无臂构造的优点在于能最大限度减小导电路径的长度,因为酶外部的导电路径部分可能是不应有的噪声、电阻或电容的源头。上面关于与臂接线的情况的考虑一般也适用于无臂构造的特殊情况、以及与直接酶耦合结合的单臂构造的特殊情况。具体而言,在没有臂的实施例中,酶仍可通过内部结构或在构象变化点处接线。
对于包括作为基本导电路径的直连酶的传感器,可通过多种环境因素增强其信号性能。例如,可调整缓冲液的选择、缓冲液添加剂,温度和施加的电压,以改善信号质量。尤其是,由于酶可与调节其动力特性的各种辅助因子复合,并且缓冲液中的盐度也像温度一样影响酶动力特性,因此可利用这些因素来改善信号传导。另外,缓冲溶液的总离子强度限定溶液中的德拜长度(即,电场在溶液中延伸的距离),并且可能因酶和底物中的电荷分布而影响载流子穿过酶的能力,因此缓冲液离子强度或总盐浓度是影响或增强信号传导的另一种手段。在利用聚合酶的实施例中,已知的是缓冲液的二价阳离子含量会影响酶的活性,并且可优化二价阳离子的选择(例如选自Mg++、Mn++、Ni++、Zn++、Co++、Ca++、Cr++)以及它们的浓度,以改善连接为基本导电路径的聚合酶的信号传导。
可优化施加的驱动电压,以改善连接为基本导电路径的酶的信号传导。根据酶内的能垒,某些电压可能改善信号传导性能。除了施加的电压之外,多个实施例还可具有栅电极(例如图3中所示的下部底物下方的埋栅),从而施加至栅电极的电压进一步调节酶电路的信号传导特性。一些实施例可采用电压谱法,其中驱动电压或栅极电压扫过一定范围的值,并且所关注的信号在该扫描的响应中,该响应包含关于酶与其底物之间的相互作用的信息。
本公开的分子电路传感器一般包括具有至少两个电接触点的酶的接线,从而使酶成为基本导电路径。这与图2的构造相反。酶的两点接线可与接合物125结合到一个或多个分子臂127上,如图10和11所示。在这些实施例中,可驱动电流穿过所述酶以进行感测,并且所述酶还可调节流过另一条分子线的电流,作为附加的感测模式。在图10中,这些导电模式由单个电极对130监视,并且结合以产生单个电流,而在图11中,这两个导电模式可由两个独立的电极对(第一电极对135和第二电极对140)监视,产生两个电流测量值145。在一些实施例中,所述传感器可包括与两个或更多接触点连接起来作为导电路径的酶126,并结合有其他传感器构造特征。将酶在两个点与输入和输出电触点连接可增强信号传导。在图10和11中示出了其他可能且非限制性的构造。
在多个实施例中,分子电路传感器包括聚合酶。图12示出了有代表性的聚合酶150,即,大肠杆菌DNA聚合酶I的Klenow片段。图12从两个不同的角度示出了酶结构的带状图,其中酶与双链DNA模板170接合。所述酶的主结构是由605个氨基酸组成的单个氨基酸序列。所述酶的二级结构和三级结构如图12所示。该图示出了酶的结构元件。尤其是,存在28个不同的α螺旋元件160和两个主要的β褶板元件165,它们由短柔性环片段155隔开。通常,这种结构元件也类似地存在于其他类型的聚合酶和其他酶蛋白中。在酶活跃的过程中,这些结构特征会参与电气、化学、机械和构象扰动,在电路内与这些特征的接线会将这些扰动转换为测量信号。
图13示出了一个实施例,其中聚合酶177被接线为基本导电路径,具体而言,聚合酶177被接线至穿过酶中心的长α螺旋180的端部。选择该α螺旋是因为它非常靠近聚合酶的活性袋,因此当它结合至引物链并通过纳入dNTP来延长引物时,能增强聚合酶活性的电流感测。结构中的其他α螺旋会提供其他感测机会,并且其他实施例包括与这种α螺旋结构、β褶板结构、或以串联形式出现的其他结构的接线。图13中所示的臂175可包括以马来酰亚胺终止的双链寡核苷酸,该马来酰亚胺与经基因工程改造到聚合酶的突变形式的精确位置的半胱氨酸耦合。在另一个实施例中,所述臂包含以马来酰亚胺封端的蛋白质α螺旋,该马来酰亚胺与这种半胱氨酸耦合。这种突变型聚合酶的一个特定实施例包括位于图3所示的接合点的半胱氨酸(C),该半胱氨酸(C)是通过以C替换氨基酸位置548的谷氨酰胺(Q)并以C替换氨基酸位置508的丝氨酸(S)而产生的,并且这两个位置恰好位于从氨基酸位置514延伸至547的一个长α螺旋(37个氨基酸)的之外并封住该长α螺旋。在一些实施例中,该突变型聚合酶的氨基酸位置584处的单个原生C被非半胱氨酸(例如S)取代,从而通过众所周知的马来酰亚胺-半胱氨酸接合方式恰好提供用于耦合由马来酰亚胺封端的臂的两个位点。为了引入半胱氨酸而进行这种氨基酸取代的执行方式应不改变高度保守的氨基酸(与其他聚合酶相比是保守的)、不改变作为接线目标物的α螺旋或其他结构元件中的氨基酸、不改变直接参与关键酶功能的氨基酸,例如与底物结合袋、DNA底物或dNTP底物结构直接相互作用的氨基酸。类似的选择原则也适用于在半胱氨酸中突变为马来酰亚胺接合点时的其他酶。
图14示出了一个替代实施例,其中图13的突变型聚合酶177直接接合185至电极187,耦合至内部α螺旋,而不使用连接臂。这种耦合例如可利用金电极和带有马来酰亚胺封端的金结合肽(GBP)来实现,以使马来酰亚胺将GBP在上述的半胱氨酸位点接合至突变型聚合酶,并且GBP接合至金电极,从而通过这两个半胱氨酸位点在聚合酶中接线。使用具有结合至聚合酶上的半胱氨酸的X-马来酰亚胺形式的接合基团,能够实现以马来酰亚胺中介的直接接合至电极的其他实施例,其中X是随后结合至电极表面的基团。
图15示出了一个替代实施例,其中使用连接臂210通过邻近聚合酶的结合凹槽205的α螺旋200将图13的突变型聚合酶177接线至电极212。该连接臂接线至聚合酶上的两个点215。在一些实施例中,这两个点具有相对于彼此移动的能力,从而实现改变电导率和增强信号传导。
图16示出了使用多个臂190将聚合酶195连接为基本导电路径并稳定其相对于电极和底物的位置和取向的一个实施例。根据多个实施例,所示的下臂对可以是导电的或绝缘的。
在多个实施例中,一种电路包括连接为基本导电路径的酶。该电路可包括第一和第二接线点,这些接线点连接至第一和第二电极,例如正电极和负电极。
在多个实施例中,该电路还可包括至少一个具有两个端部的臂分子,其中一端与酶结合,而另一端与所述电极中的至少一个结合,其中所述至少一个臂分子作为所述酶分子与所述电极中的至少一个之间的电线。这种臂分子可选自由双链寡核苷酸、肽核酸(PNA)双链体、PNA-DNA杂化双链体、蛋白质α螺旋、类似石墨烯的纳米带、天然聚合物、合成有机分子(例如合成聚合物)、以及抗体Fab结构域组成的组。在其他示例中,所述酶直接接线至电极,而不使用任何臂分子。所述接线可以是连接至酶中的内部结构元件,例如α螺旋或β褶板,或串联的多个此类元件。
在多个实施例中,一种电路包括在发生相对构象变化的点连接的酶。在一些方面中,臂包括具有取决于张力的电导率的分子。在其他示例中,臂分子可具有取决于扭转或扭曲的电导率。可使用附加的接线点将酶耦合在附加位点。
在多个实施例中,一种电路包括聚合酶,例如大肠杆菌Pol I Klenow片段,其中所述接线是连接至在位置514和547的氨基酸之间延伸的主α螺旋。这种连接可能需要将基因工程半胱氨酸置于这些氨基酸位置处或附近。可使用包括聚合酶的电路从由聚合酶处理的DNA模板感测序列信息。
本公开的多个实施例的电路可暴露于包含引物单链DNA和/或dNTP的溶液,其中在聚合酶接合并延长模板时测量流过电路的电流,并对得到的信号进行处理,以识别提供关于由聚合酶处理的DNA分子的基础序列信息的特征。
酶分子与正电极和负电极中的至少一个之间的连接可包括以下结构中的任何一种:天然半胱氨酸、基因工程半胱氨酸、具有接合残基的基因工程氨基酸、或者包括具有接合配偶的肽的基因工程肽结构域。在一些方面中,所述接线是连接至酶的拇指和手指结构域上的点,其中这些点随着聚合酶处理DNA模板而发生超过1纳米的相对运动。在其他方面中,所述聚合酶被改造为具有延长结构域,该延长结构域随着聚合酶处理DNA模板而产生更大范围的相对运动。例如,可通过延长酶中的各种结构域来加强酶的构象变化。聚合酶还可改造为具有额外的电荷基团,这些电荷基团随着所述酶处理DNA模板而可变地影响内部导电路径。
在多个实施例中,电路暴露于包含经过修饰的dNTP的溶液,所述经过修饰的dNTP随着酶处理DNA或RNA模板而可变地影响内部导电路径。在某些情况下,所述聚合酶是大肠杆菌Pol I聚合酶、Bst聚合酶、Taq聚合酶、Phi29聚合酶、T7聚合酶和逆转录酶之一的基因修饰形式。在其他示例中,电路暴露于以下条件中的一种或多种:离子强度降低的缓冲液、包括经过修饰的dNTP的缓冲液、包括变化的二价阳离子浓度的缓冲液、施加在主电极上的特定电压、栅电极电压、或施加在主电极或栅电极上的电压谱或扫描。
在多个实施例中,所述聚合酶包括能够直接作用于RNA模板的逆转录酶或经过基因修饰的逆转录酶。在这些电路中使用逆转录酶的益处在于,逆转录酶能直接处理RNA模板,因此提供了一种对RNA分子进行直接测序的方法。在多个方面中,所述逆转录酶可以是任何单体逆转录酶或其遗传修饰形式,例如莫洛尼鼠白血病病毒逆转录酶、猪内源性逆转录病毒逆转录酶、牛白血病病毒逆转录酶、小鼠乳腺肿瘤病毒逆转录酶、或异二聚体逆转录酶,例如人免疫缺陷病毒逆转录酶或劳斯肉瘤病毒逆转录酶。
在一些示例中,公开了一种对DNA分子进行测序的方法。所述方法包括:提供基于酶的电路,该电路具有隔开的正电极和负电极、以及连接至正电极和负电极连接从而在这些电极之间形成导电通路的聚合酶分子;引发通过所述电路的电压或电流中的至少一种;使所述电路暴露于含有引物单链DNA和/或dNTP的溶液;以及在聚合酶接合并延长模板时,测量通过所述电路的电信号,其中对所述电信号进行处理以识别提供关于由聚合酶处理的DNA分子的基础序列信息的特征。
在其他方面中,公开了一种分子检测方法。所述方法包括:(a)提供基于酶的电路,该电路具有间隔开的正电极和负电极、以及连接至正电极和负电极从而在这些电极与栅电极之间形成导电通路的聚合酶分子;(b)引发通过所述电路的电压或电流中的至少一种;(c)使所述电路暴露于以下条件中的至少一种:离子强度降低的缓冲液、包括经过修饰的dNTP的缓冲液、包括变化的二价阳离子浓度的缓冲液、施加在主电极上的特定电压、栅电极电压、或施加在主电极或栅电极上的电压谱或扫描;以及(d)测量所述电路中的电气变化。
本发明提供了一种基于酶的分子传感器及其制造和使用方法。对“多个实施例”、“单个实施例”、“一个实施例”、“示例性实施例”等的引用表示所述的实施例可包括特定的特征、结构或特性,但是不一定每个实施例都包括该特定特征、结构或特征。而且,这样的短语不一定指代同一个实施例。此外,在结合实施例说明特定特征、结构或特性时,应认识到,在本领域技术人员的知识范围内,此类特征、结构或特性可结合其他实施例实施,不论这些实施例在本文中是否已明确说明。在阅读说明书之后,如何在替代实施例中实现本公开对于相关领域的技术人员来说将是显而易见的。
本发明的益处、其他优点和问题的解决方案在上文中是参照具体实施例说明的。但是,这些益处、优点、问题的解决方案、以及可能带来任何益处、优点或解决方案或使其变得更加明显的任何元素不应理解为是本公开的关键、必要或本质特征或元素。因此,本公开的范围仅受所附权利要求的限制,在所附权利要求中,除非另行明确声明,否则对单数形式的元件的引用并不旨在表示“一个且仅有一个”,而是表示“一个或多个”。此外,在权利要求或说明书中使用类似于“A、B和C中的至少一个”或“A、B或C中的至少一个”的短语时,应将该短语解读为指在一个实施例中可单独存在A、在一个实施例中可单独存在B、在一个实施例中可单独存在C、或者在单个实施例中可存在元素A、B和C的任何组合;例如A和B、A和C、B和C或A和B和C。
本领域普通技术人员已知的上述各个实施例的元素的所有结构、化学和功能等同物通过引用明确地结合在本文中,并且意图被本权利要求所涵盖。而且,装置或方法不一定必须解决本公开寻求解决的每个问题,因为这些问题被权利要求所涵盖。此外,本公开中的任何元件、部件或方法步骤都不意图贡献给公众,无论在权利要求中是否明确列举了该元件、部件或方法步骤。任何要求保护的元素都不意图引用35U.S.C.,112(f),除非使用短语“用于......的装置”明确叙述该元素。在本文中所使用的术语“包括”、“包含”或其任何其他变化形式意图涵盖非排他性的包含,从而包括一系列元素的分子、组成、过程、方法或装置不仅包含这些元素,还可包含未明确列出或此类分子、组成、过程、方法或装置所固有的其他元素。
Claims (20)
1.一种电路,包括:
正电极;
与所述正电极隔开的负电极;和
连接至所述正电极和所述负电极两者从而在所述正电极与所述负电极之间形成导电通路的酶。
2.根据权利要求1所述的电路,其中所述酶包括连接至所述正电极的第一接线点和连接至所述负电极的第二接线点。
3.根据权利要求1所述的电路,还包括具有第一端和第二端的至少一个臂分子,所述第一端结合至所述酶,所述第二端结合至所述电极中的至少一个电极,其中所述至少一个臂分子用作所述酶与所述电极中的至少一个电极之间的电线。
4.根据权利要求3所述的电路,其中所述至少一个臂分子选自由双链寡核苷酸、肽核酸双链体、肽核酸-DNA杂化双链体、蛋白质α螺旋、类似石墨烯的纳米带、天然聚合物、合成聚合物和抗体Fab结构域组成的组。
5.根据权利要求1所述的电路,其中所述电极中的至少一个电极连接至所述酶的内部结构元件。
6.根据权利要求5所述的电路,其中所述内部结构元件选自由α螺旋、β褶板和串联的多个这种元件组成的组。
7.根据权利要求1所述的电路,其中所述电极中的至少一个电极在所述酶的能够发生构象变化的位置处连接至所述酶。
8.根据权利要求3所述的电路,其中所述至少一个臂包括具有取决于张拉、扭曲或扭转的电导率的分子。
9.根据权利要求1所述的电路,其中所述酶包括聚合酶。
10.根据权利要求9的电路,其中所述聚合酶包括大肠杆菌PolI Klenow片段。
11.根据权利要求10所述的电路,其中所述正电极和所述负电极各自在位置514和547的氨基酸之间延伸的所述聚合酶的主α螺旋内的连接点处连接至所述聚合酶。
12.一种包括如权利要求9所述的电路的分子传感器,其中所述传感器能够用于从由所述聚合酶处理的DNA模板感测序列信息。
13.根据权利要求9所述的电路,其中所述正电极和所述负电极各自在所述聚合酶的拇指和手指结构域上的连接点处连接至所述聚合酶,并且其中在所述聚合酶处理DNA模板时,这些点发生超过1纳米的相对运动。
14.根据权利要求13所述的电路,其中所述聚合酶被改造为具有延长结构域,所述延长结构域在所述聚合酶处理所述DNA模板时产生更大范围的相对运动。
15.根据权利要求9所述的电路,其中所述聚合酶被改造为具有附加的电荷基团,这些附加的电荷基团在所述酶处理DNA模板时可变地影响内部导电通路。
16.根据权利要求9所述的电路,其中所述聚合酶是大肠杆菌Pol I聚合酶、Bst聚合酶、Taq聚合酶、Phi29聚合酶、T7聚合酶或逆转录酶的基因修饰形式。
17.根据权利要求1所述的电路,其中所述正电极和所述负电极各自在所述酶内的连接点处连接至所述酶,所述连接点包括天然半胱氨酸、基因工程半胱氨酸、具有接合残基的基因工程氨基酸、或包括具有接合配偶的肽的基因工程肽结构域中的至少一种。
18.根据权利要求9所述的电路,还包括栅电极。
19.一种对DNA分子进行测序的方法,包括:
提供如权利要求9所述的电路;
引发通过所述电路的电压或电流中的至少一种;
使所述电路暴露于含有引物单链DNA和/或dNTP的溶液;和
当聚合酶接合并延长模板时,测量通过所述电路的电信号,
其中对所述电信号进行处理以识别提供关于由所述聚合酶处理的所述DNA分子的基础序列的信息的特征。
20.一种分子检测方法,包括:
提供如权利要求18所述的电路;
引发通过所述电路的电压或电流中的至少一种;
使所述电路暴露于以下条件中的至少一种:离子强度降低的缓冲液、包括经过修饰的dNTP的缓冲液、包括变化的二价阳离子浓度的缓冲液、施加在主电极上的特定电压、栅电极电压、或施加在主电极或栅电极上的电压谱或扫描;和
测量所述电路中的电气变化。
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