CN111705044B - 新型可控高活性g四链体dna酶的构建 - Google Patents

新型可控高活性g四链体dna酶的构建 Download PDF

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CN111705044B
CN111705044B CN202010482177.5A CN202010482177A CN111705044B CN 111705044 B CN111705044 B CN 111705044B CN 202010482177 A CN202010482177 A CN 202010482177A CN 111705044 B CN111705044 B CN 111705044B
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quadruplex
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dnase
cytosine
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CN111705044A (zh
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周俊
鞠熀先
陈杰林
程明攀
王佳伟
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Nanjing University
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Abstract

本发明涉及一种间位胞嘧啶增强并利用单碱基修饰技术进一步实现高催化活性G四链体DNA酶的构建。首先,在富含G碱基DNA序列的3’端修饰胞嘧啶及其衍生物,在含有100mM K离子的缓冲中,95℃退火5min,形成G四链体结构。然后将该G四链体与血红素混合,形成DNA酶。通过测试不同H2O2浓度下DNA酶的催化活性并利用米氏模型进行拟合、计算获得不同G四链体DNA酶的米氏常数。最终,当3’端间位碱基是氮杂胞苷时,其活性与天然蛋白酶HRP的活性达到同个数量级(kcat(F3T‑azaC)=22.7s‑1)。该高活性G四链体DNA酶设计简单,制备快速,价格低廉,便于储存,具有一定的应用前景。

Description

新型可控高活性G四链体DNA酶的构建
一、技术领域
本发明为一种间位胞嘧啶增强催化活性且可通过胞嘧啶修饰实现新型高催化活性G四链体DNA酶的构建及其制备方法。
二、背景技术
天然生物催化剂由于其易受环境干扰,作用机制复杂而广受诟病。设计、构建结构简单、催化活性高的仿生酶具有重要意义。目前,人工构建的纳米仿生酶大都集中于纳米粒子,如Fe3O4等。这类纳米仿生酶具有更优的抗环境干扰能力,但仍受限于合成复杂、重复性有限等缺点。G四链体/血红素(hemin)DNA酶因其易于合成、成本低、设计性强、稳定性高等优点,广泛应用于各种纳米领域。然而,DNA酶仍存在活性低的缺点。因此,外源性激活因子或DNA序列修饰策略被应用于提高其催化活性。虽几经努力,DNA酶活性依旧无法与天然酶相媲美。富G序列形成的G平面为hemin提供了一个良好的平台,而空间邻近的胞嘧啶由于其较小的嘧啶环以及环上羰基的存在,导致其激活能力一直不理想。
本发明在富含G碱基的DNA序列末端修饰间位胞嘧啶,在钾离子辅助下形成分子内平行G四链体,与hemin结合形成了G四链体DNA酶。间位上的胞嘧啶能够显著提高DNA酶的催化活性,进一步对该胞嘧啶进行修饰,可以使之达到与天然酶同数量级的催化活性。这种DNA酶合成方法简单、催化活性高、便于保存,在工业应用上具有很大的应用前景。
三、发明内容
本发明的目的是:利用富电子胞嘧啶衍生物修饰的富G碱基DNA序列,结合血红素(hemin),使胞嘧啶衍生物与hemin-H2O2形成更为稳定的催化活性中间体,开发一种可控的新型人工模拟酶(如图1所示),实现高活性G四链体DNA酶的构建。
平行G四链体有利于血红素的结合,稳定性强且活性高。实验发现,间位嘧啶可以较好地与血红素进行配位,较大程度地增强DNA酶的活力(kcat(F3TC)=1.72s-1 )。如图1所示,我们通过将不同基团修饰于间位胞嘧啶上,发现富电子基团修饰的间位胞嘧啶极大地增强了G四链体DNA酶的活性(kcat(F3T-azaC)=22.7s-1 ),对于DNA酶在生物传感器以及细胞内靶标识别的应用与发展具有重要意义。
本发明通过以下技术方案来实现:
(1)如图1所示,在含有四段连续3个G碱基的DNA单链3’端修饰胞嘧啶碱基,在含100mM钾离子的缓冲液中95℃加热5分钟,冷却至室温,形成分子内G四链体结构。
(2)将形成的G四链体和hemin混合,在室温下温育0.5小时,形成高活性G四链体DNA酶。
(3)如图1所示,通过改变胞嘧啶的修饰基团,调节其嘧啶环上的电荷密度,利用DNA酶催化H2O2-ABTS(2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸)反应,用分光光度法监测其不同碱基突变下的催化活性。
与现有技术相比,本发明具有以下特点:
本发明将不同胞嘧啶衍生物修饰在富G碱基序列的末端,形成的G四链体结合hemin构建形成G四链体DNA酶,间位上的胞嘧啶能够显著提高DNA酶的催化活性,同时修饰后的基团为胞嘧啶提供电荷,进一步激活DNA酶的催化活力。相对于现有DNA酶,具有以下特点:
(1)该DNA酶具有合成过程简单、价格低廉、易于保存、激活位点明晰的优点。
(2)该DNA酶在间位胞嘧啶的激活下具有高的催化活性,修饰之后可以进一步提高活性,达到与天然酶相同数量级的酶活性。
(3)如图1所示,间位胞嘧啶可以在常温下达到kcat=1.72s-1的催化活性;氮杂胞苷的修饰可以进一步提高了催化活性,达到kcat=22.7s-1,该活性与天然酶处于同个数量级。
四、附图说明
图1.间位修饰胞嘧啶衍生物的分子内G四链体及其DNA酶形成与催化示意图。
五、具体实施方式
实施例1:结合附图1,说明分子内G四链体结构的形成
3’端修饰胞嘧啶的富G碱基DNA序列,在含有100mM钾离子的Britton-Robinson(B-R)缓冲液中(10mM缓冲液,pH 7.0)95℃加热5分钟后,缓慢退火至室温,形成分子内G四链体结构。
实施例2:结合附图1,利用圆二色光谱,热差谱等技术说明分子内G四链体的形成,与hemin结合成DNA酶后,利用分光光度计测试其催化H2O2-ABTS的反应速率。
(1)酶溶液的制备:将形成的分子内G四链体结构和hemin混合在含100mM K+,0.05%Triton X-100和1%DMSO的10mM B-R缓冲液(pH 7.0)中,最终浓度分别为0.4μM和0.8μM,室温孵育0.5小时。
(2)控制酶溶液的温度在25℃之间,加入0.6mM ABTS,然后用0.1-10mM H2O2触发反应,用分光光度法监测其酶反应动力学1min,获得其反应初始速率之后,利用米氏模型进行拟合,获得其米氏常数,研究酶的催化活性。

Claims (2)

1.一种新型可控高活性G四链体DNA酶,通过修饰间位胞嘧啶或氮杂胞苷以实现G四链体DNA酶的高催化活性;其特征在于所述DNA酶是利用单端修饰间位胞嘧啶碱基或氮杂胞苷的富G碱基DNA序列在含100mM钾离子的缓冲液中,95℃加热5min,退火冷却形成分子内G四链体结构,与血红素结合而形成高活性DNA酶。
2.根据权利要求1所述的DNA酶,室温下,在间位胞嘧啶存在时,催化活性从kcat=0.305s-1提高至kcat=1.72s-1;修饰氮杂胞苷时,其催化活性进一步激活至kcat=22.7s-1,与天然酶活性处于同一数量级。
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