基于G-四链体探针和链置换放大的光学分析新方法
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摘要
核酸是一种广泛存在于生物体内的生物大分子，为生命的最基本物质之一。伴随着体外合成技术，特别是固相合成的出现，它已成为一种重要的分子工具，在生化分析领域扮演着重要的角色。由于核酸具有易于合成、稳定性好、生物相容性好、设计简单、信号机制灵活等诸多优势，它被广泛应用于分子探针、分子机器、生物传感器的构建，另外它可与各种工具酶联用开发高效快速的信号放大方法。目前，基于核酸的分子探针和放大方法已成为构建光化学生物传感器的重要元件。这类传感器具有灵敏度高、操作简便、分析速度快、检测成本低、易于实现高通量和微型化等优点，在生物化学、生物医学、环境监测、食品、医药和军事等领域有着极其广阔的应用前景。新型多功能核酸探针的开发及高效信号放大方法的设计对发展多功能、高灵敏的光学生物传感器至关重要。基于以上考虑，综合文献报道，本文主要在开发新型多功能核酸探针和解决当前链置换放大方法在使用过程中存在的焦点和难点问题做了一些工作，主要内容如下：
鉴于含连续不间断富G片段的DNA探针具有优越的自组装性能已在生物材料领域引起广泛关注，而在生物传感器领域的报道相对较少，在第2章中，我们以一条含7个连续G碱基(G7s)的富G荧光探针为模型，对该类富G探针的自组装性能和荧光光学性质进行了详细研究。通过圆二色谱（CD）、紫外光谱（UV）和电泳分析发现该富G探针主要自组装形成分子间平行G-四链体结构（intermolecular parallel G-quadruplex，IGQ)。伴随这种独特的自组装行为，该探针展现出了独特的荧光猝灭机理，荧光背景非常低，并且猝灭信号可以通过加入互补序列进行调控。荧光信号恢复可达近90倍，可与传统的分子信标媲美。实验结果表明该类IGQ信号探针在光学传感器领域具有良好的应用前景。
第3章我们将IGQ信号探针与无标记的发夹探针结合，开发了一种新的无猝灭标记的分子信标（IGQ-MB），应用于人P53基因检测。得益于IGQ信号探针独特的光学性质，我们实现了MB的识别序列与信号报告基团的分离。因此，针对不同的目标，我们只需根据需求重新合成无标记的发夹序列，从而大大降低了MB的实际操作成本，增强了通用性。另外，发夹探针末端的无标记有利于其在聚合反应以及界面分析方法中的应用，拓宽了MB的适用范围。G-四链体结构的引入使得该设计在疾病诊断和药物运载领域具有更广阔的前景。
在采用荧光嵌入剂作为信号报告基团的链置换放大(SDA)反应体系中，不依赖模板/引物的非特异性扩增现象广泛存在，并严重严重影响检测体系的背景信号。在第4章中，我们发现向体系中加入一种蛋白质能有效地抑制这种非特异性扩增。基于该发现我们进一步提出了一种新的SDA模型——切刻反转式SDA模型。该模型主要基于一条一体化的模板探针，告别了传统方法使用多条引物探针和循环高温加热操作的模式，不仅操作简单价格低廉，而且适用于37℃条件下DNA及蛋白质的无标记检测。
第5章我们通过实验指出并证明了在传统的采用目标识别和信号传导相分离的链置换扩增放大检测方法中，存在“信号耗损”现象。针对该现象我们提出了一种新型的基于集成式信号读出方式的一体化探针分子机器，用于可卡因的定量分析。通过采用目标识别与信号传导一体化的设计，巧妙避免了“信号耗损”，缩短了分子机器的信号响应时间，提高了的检测灵敏度，同时扩宽了响应范围近500倍。
第6章描述了一种摆臂式DNA(LPOD)纳米开关，并将其应用于目标DNA序列的特异性识别以及高灵敏定量检测。逐步加入启动探针或制动探针与开关杂交，能使该开关的单链手臂在9.4nm范围内进行摆臂运动，调控FRET反应，实现开关的反复开合。与传统的“镊子”型的纳米开关相比，该开关只有一个手臂呈现单链状态，其它均呈刚性双链结构，一方面有效地避免了纳米开关之间的共杂交，另一方面降低了DNA纳米器件的不可预知形变几率。另外，该开关采用单一的启动结合位点，有效地避免了间隔碱基片段的拉伸作用，更利于启动探针的杂交反应。
Nucleic acids are important biological macromolecules essential for life, which arefound in abundance in all living things. As the development of synthesistechnology in vitro, especially with the discovery of the solid phase synthesis,nucleic acids have become important molecular tools and play important roles inbiochemical analysis. With the advantages of stable, biocompatible, simple design,easy to synthesize and flexible signal mechanism, nucleic acids have been widelyapplied in designing molecular probe, constructing molecular machines andbiosensors. Additionally, by combining with various tool enzymes, they can also beused in developing efficient signal amplification strategies. Nowadays, nucleic acidsbased molecular probe and signal amplification strategy have become importantelements of photochemical biosensor. This kind of biosensors has wide applicationprospects in chemistry, biomedicine, environmental monitoring, food industry,medicine and military affairs, attributing to its high sensitivity, easy operation, fastresponse, low cost and easy to realize high throughput analysis and miniaturization.Therefore, developing nucleic acid probe with multifunctions and efficient signalamplification strategy is vital for photochemical biosensor with excellent properties.We focused on these points and developed several photochemical biosensors in thispaper. The details are summarized as follows:
(1) In chapter2, single fluorescein (FAM)-labeled G-rich signaling probewithout any quencher is developed as the model. This probe was designed to possessa continuous seven-guanine fragment (G7s) and a random tail. Its intermolecularparallel G-quadruplex (IGQ) structure is validated by recording fluorescenceemission, CD, UV, and gel electropherogram. Moreover, the developed probepossesses low self-quenching-based background fluorescence, and the restoration ofquenched fluorescence can be easily accomplished in a simple manner. Excitingly,more than90-fold fluorescence enhancement can be detected for an improvedIGQ-structure-based probe after hybridization to complementary strand, dramaticallyhigher than signal of traditional molecular beacon. The solid data imply thefascinating potential application of the concept of single fluorophore-labeled G-richprobe in biosensor development and G-quadruplex researches to acquire the furtherinformation on the function of vital nucleic acids in biological processes.
(2) In chapter3, we further applied IGQ signaling probe to bind with an unmodified hairpin sequence and fabricated a novel quencher free molecular beacon(called intermolecular G-quadruplexb-based molecular beacon, IGQ-MB) for detectingof human P53gene. In this novel strategy, the molecular recognition element (hairpinsequence) and signal reporter has been successfully separated. Therefore, in practicalapplication, users just need to resynthesis the unmodified hairpin sequences accordingto the various requirements in optimization of MB or in detection of different targets,which obviously reduce the cost and improve the convenience. The novel strategy hasgreat potential in disease diagnosis, drug discovery and solid surface analysis.
(3) There is a template/primer independent non-specific reaction existing in moststrand dispalsment amplification (SDA)-based detection systems, especially for theone with intercalating dye as the signal reporter, which seriously interference thebackground signal of the detection system. In Chapter4, we found that suchnon-specific reaction can be effectively inhibited by adding a special protein into thesystem. Furthermore, a novel SDA-based strategy, called nicking-induced reversingSDA model, is developed as a proof-of-concept. The model is mainly based on anintegrated template probe which is distinguished from tranditional multiprobe basedSDA system. The novel label-free SDA-based strategy is not only simple andinexpensive, but also suitable for detection of DNA and protein under the conditionof37°C.
(4) In chapter5, we pointed out and demonstrated that there is a “signalmisreading” behavior in existing autonomous machines where the target recognitionprocess and signal transduction is separated from each other. We further developed aintegrated signal transduction-based autonomous aptameric machine, in which therecognition element and signal reporters are integrated into a DNA strand. This newmachine can execute the in situ amplification of target-induced signal. The authenticoperation behavior of autonomous DNA machine is discovered: the machine’s productsdirectly hybridize to the “track” rather than to the signaling probes. Along this line, themachine is employed to detect the cocaine in a more straightforward fashion, andimproved assay characteristics (for example, the dynamic response range is widenedby more than500-fold) are achieved. Our efforts not only clarify the concept describedin traditional autonomous DNA machines but also have made technologicaladvancements that are expected to be especially valuable in designing nucleicacid-based machines employed in basic research and medical diagnosis.
(5) In chapter6, a powerful pendulum-type DNA (LPOD) nanoswitch, which canperform a reversible on/off molecular motion at an about9.4-nm scale, is developed as a proof-of-concept, and the sequence-specific recognition and sensitive quantificationof target olignucleotides are demonstrated utilizing this screening scheme. In contrastto the existing nanomachines (e.g. tweezers), the hybridization of the fuel strand withthe nanodevice opens the hairpin structure and straightens one arm, bringing thequencher into the close proximity of the fluorophore for FRET. Additionally, thepresent scheme has eliminated dimers as only one arm is kept in the single-strandedstate avoiding the hybridization of nanodevices with each other. Furthermore, ashybridization reaction occurring at a single “binding site” rather than the dual “bindingsites” eliminates the effect of spacer segments that can encountered for an integratedsignaling probe, the nanomachine seems to exhibit significant advantages.
鉴于含连续不间断富G片段的DNA探针具有优越的自组装性能已在生物材料领域引起广泛关注，而在生物传感器领域的报道相对较少，在第2章中，我们以一条含7个连续G碱基(G7s)的富G荧光探针为模型，对该类富G探针的自组装性能和荧光光学性质进行了详细研究。通过圆二色谱（CD）、紫外光谱（UV）和电泳分析发现该富G探针主要自组装形成分子间平行G-四链体结构（intermolecular parallel G-quadruplex，IGQ)。伴随这种独特的自组装行为，该探针展现出了独特的荧光猝灭机理，荧光背景非常低，并且猝灭信号可以通过加入互补序列进行调控。荧光信号恢复可达近90倍，可与传统的分子信标媲美。实验结果表明该类IGQ信号探针在光学传感器领域具有良好的应用前景。
第3章我们将IGQ信号探针与无标记的发夹探针结合，开发了一种新的无猝灭标记的分子信标（IGQ-MB），应用于人P53基因检测。得益于IGQ信号探针独特的光学性质，我们实现了MB的识别序列与信号报告基团的分离。因此，针对不同的目标，我们只需根据需求重新合成无标记的发夹序列，从而大大降低了MB的实际操作成本，增强了通用性。另外，发夹探针末端的无标记有利于其在聚合反应以及界面分析方法中的应用，拓宽了MB的适用范围。G-四链体结构的引入使得该设计在疾病诊断和药物运载领域具有更广阔的前景。
在采用荧光嵌入剂作为信号报告基团的链置换放大(SDA)反应体系中，不依赖模板/引物的非特异性扩增现象广泛存在，并严重严重影响检测体系的背景信号。在第4章中，我们发现向体系中加入一种蛋白质能有效地抑制这种非特异性扩增。基于该发现我们进一步提出了一种新的SDA模型——切刻反转式SDA模型。该模型主要基于一条一体化的模板探针，告别了传统方法使用多条引物探针和循环高温加热操作的模式，不仅操作简单价格低廉，而且适用于37℃条件下DNA及蛋白质的无标记检测。
第5章我们通过实验指出并证明了在传统的采用目标识别和信号传导相分离的链置换扩增放大检测方法中，存在“信号耗损”现象。针对该现象我们提出了一种新型的基于集成式信号读出方式的一体化探针分子机器，用于可卡因的定量分析。通过采用目标识别与信号传导一体化的设计，巧妙避免了“信号耗损”，缩短了分子机器的信号响应时间，提高了的检测灵敏度，同时扩宽了响应范围近500倍。
第6章描述了一种摆臂式DNA(LPOD)纳米开关，并将其应用于目标DNA序列的特异性识别以及高灵敏定量检测。逐步加入启动探针或制动探针与开关杂交，能使该开关的单链手臂在9.4nm范围内进行摆臂运动，调控FRET反应，实现开关的反复开合。与传统的“镊子”型的纳米开关相比，该开关只有一个手臂呈现单链状态，其它均呈刚性双链结构，一方面有效地避免了纳米开关之间的共杂交，另一方面降低了DNA纳米器件的不可预知形变几率。另外，该开关采用单一的启动结合位点，有效地避免了间隔碱基片段的拉伸作用，更利于启动探针的杂交反应。
Nucleic acids are important biological macromolecules essential for life, which arefound in abundance in all living things. As the development of synthesistechnology in vitro, especially with the discovery of the solid phase synthesis,nucleic acids have become important molecular tools and play important roles inbiochemical analysis. With the advantages of stable, biocompatible, simple design,easy to synthesize and flexible signal mechanism, nucleic acids have been widelyapplied in designing molecular probe, constructing molecular machines andbiosensors. Additionally, by combining with various tool enzymes, they can also beused in developing efficient signal amplification strategies. Nowadays, nucleic acidsbased molecular probe and signal amplification strategy have become importantelements of photochemical biosensor. This kind of biosensors has wide applicationprospects in chemistry, biomedicine, environmental monitoring, food industry,medicine and military affairs, attributing to its high sensitivity, easy operation, fastresponse, low cost and easy to realize high throughput analysis and miniaturization.Therefore, developing nucleic acid probe with multifunctions and efficient signalamplification strategy is vital for photochemical biosensor with excellent properties.We focused on these points and developed several photochemical biosensors in thispaper. The details are summarized as follows:
(1) In chapter2, single fluorescein (FAM)-labeled G-rich signaling probewithout any quencher is developed as the model. This probe was designed to possessa continuous seven-guanine fragment (G7s) and a random tail. Its intermolecularparallel G-quadruplex (IGQ) structure is validated by recording fluorescenceemission, CD, UV, and gel electropherogram. Moreover, the developed probepossesses low self-quenching-based background fluorescence, and the restoration ofquenched fluorescence can be easily accomplished in a simple manner. Excitingly,more than90-fold fluorescence enhancement can be detected for an improvedIGQ-structure-based probe after hybridization to complementary strand, dramaticallyhigher than signal of traditional molecular beacon. The solid data imply thefascinating potential application of the concept of single fluorophore-labeled G-richprobe in biosensor development and G-quadruplex researches to acquire the furtherinformation on the function of vital nucleic acids in biological processes.
(2) In chapter3, we further applied IGQ signaling probe to bind with an unmodified hairpin sequence and fabricated a novel quencher free molecular beacon(called intermolecular G-quadruplexb-based molecular beacon, IGQ-MB) for detectingof human P53gene. In this novel strategy, the molecular recognition element (hairpinsequence) and signal reporter has been successfully separated. Therefore, in practicalapplication, users just need to resynthesis the unmodified hairpin sequences accordingto the various requirements in optimization of MB or in detection of different targets,which obviously reduce the cost and improve the convenience. The novel strategy hasgreat potential in disease diagnosis, drug discovery and solid surface analysis.
(3) There is a template/primer independent non-specific reaction existing in moststrand dispalsment amplification (SDA)-based detection systems, especially for theone with intercalating dye as the signal reporter, which seriously interference thebackground signal of the detection system. In Chapter4, we found that suchnon-specific reaction can be effectively inhibited by adding a special protein into thesystem. Furthermore, a novel SDA-based strategy, called nicking-induced reversingSDA model, is developed as a proof-of-concept. The model is mainly based on anintegrated template probe which is distinguished from tranditional multiprobe basedSDA system. The novel label-free SDA-based strategy is not only simple andinexpensive, but also suitable for detection of DNA and protein under the conditionof37°C.
(4) In chapter5, we pointed out and demonstrated that there is a “signalmisreading” behavior in existing autonomous machines where the target recognitionprocess and signal transduction is separated from each other. We further developed aintegrated signal transduction-based autonomous aptameric machine, in which therecognition element and signal reporters are integrated into a DNA strand. This newmachine can execute the in situ amplification of target-induced signal. The authenticoperation behavior of autonomous DNA machine is discovered: the machine’s productsdirectly hybridize to the “track” rather than to the signaling probes. Along this line, themachine is employed to detect the cocaine in a more straightforward fashion, andimproved assay characteristics (for example, the dynamic response range is widenedby more than500-fold) are achieved. Our efforts not only clarify the concept describedin traditional autonomous DNA machines but also have made technologicaladvancements that are expected to be especially valuable in designing nucleicacid-based machines employed in basic research and medical diagnosis.
(5) In chapter6, a powerful pendulum-type DNA (LPOD) nanoswitch, which canperform a reversible on/off molecular motion at an about9.4-nm scale, is developed as a proof-of-concept, and the sequence-specific recognition and sensitive quantificationof target olignucleotides are demonstrated utilizing this screening scheme. In contrastto the existing nanomachines (e.g. tweezers), the hybridization of the fuel strand withthe nanodevice opens the hairpin structure and straightens one arm, bringing thequencher into the close proximity of the fluorophore for FRET. Additionally, thepresent scheme has eliminated dimers as only one arm is kept in the single-strandedstate avoiding the hybridization of nanodevices with each other. Furthermore, ashybridization reaction occurring at a single “binding site” rather than the dual “bindingsites” eliminates the effect of spacer segments that can encountered for an integratedsignaling probe, the nanomachine seems to exhibit significant advantages.
引文
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