基于适配体的靶标检测通过 3 级 G-四链体等温指数扩增反应进行
Summary
本协议演示了使用快速，3阶段，基于适体的指数扩增测定来检测目标。涵盖样品制备、信号放大和显色，以实现该系统，以识别茶碱的存在而不是咖啡因的存在。
Abstract
核酸适配体是以高亲和力和特异性结合的靶标识别分子。可以利用这些特性来控制具有信号产生能力的其他分子。对于本文描述的系统，通过适体结构域（改良的锤头核酶的Stem II）进行目标识别，通过稳定初始非结构化结构体来激活自裂解核酶。顺式切割RNA作用于茎III和茎I的连接处，产生两种切割产物。较长的裂解产物引发两种相似催化活性G-四链体的等温指数扩增反应（EXPAR）。这些产生的扩增产物催化过氧化物酶还原，这与具有肉眼可以检测到的输出的比色底物的还原相结合。本研究中描述的三部分系统通过产生视觉可检测的信号来改进检测方式，例如酶联免疫吸附测定（ELISA），以在短短15分钟内指示低至0.5μM茶碱的存在。
Introduction
核酸适配体通常是通过进化过程选择的单链DNA或RNA，与所需靶标具有高结合亲和力和特异性1。除了结合能力外，核酸适配体还可以与具有信号输出功能2，3的基序连接和控制基序，从而放大所述信号并提高系统的灵敏度。G-四链体等温指数扩增反应（GQ-EXPAR）系统是一个由三部分组成的系统（图1），当连续组分被添加到单个反应容器中以产生视觉输出时，该系统会产生视觉信号4。该系统允许使用简化的工作流程在15分钟内检测给定样品中的特定靶标，本文中的茶碱，以允许快速，特异性地检测感兴趣的靶标。对于目标浓度的特异性定量比短时间内响应的高特异性更不关心的样品，必须考虑使用此方法。
变构核糖开关是一种经历自我切割的结构转换RNA分子（核酶），产生初始信号。该结构基于锤头核酶，在Stem II中引入了适体结构域作为裂解活性的调节剂。当其适体结构域稳定与其靶标5结合时，其自切割功能被激活。否则，交换机在其本机状态下处于非活动状态。
随后的指数扩增反应（EXPAR）使用自裂解RNA链从第一阶段释放到启动等温扩增反应6。EXPAR的扩增产物具有过氧化物酶活性7，作为系统最后阶段的基础。当某些基材与过氧化物击穿一起被氧化时，它们会产生荧光输出，可以在各种仪器上测量。可以替代其他常见的基材来生产用于视觉检测的有色产品。EXPAR及其扩增产物的过氧化物酶活性充当2级信号增强剂，与传统策略相比，将灵敏度提高到更高的水平7，8。
茶碱与咖啡因的检测被用作该检测平台特异性的一个例子，因为它们仅相差一个甲基（图2）。该系统的演示产生比色输出，用于至少500 nM茶碱的视觉检测。
Protocol
管制备见 补充表1 （反应设置表），包括反应组分的特定体积和浓度。此处演示的协议使用材料 表中描述的预组装检测平台套件。除非另有说明，否则所有组件必须保存在冰上。 1.核酶的制备 注意：主要检测成分是识别茶碱的变构（适体调节）核酶（见 补充表2）。 在PCR管中收集5U的T4多核苷酸?…
Representative Results
图1所示的检测平台可在短时间内将适体靶标识别转换为样品制备（靶标与非靶标，图2）之间的视觉差异。Soukup等人5鉴定的变构核酶作为创建噪声较小的序列的起点，该序列在对照和阴性样品上响应目标。优化后的构建体能够在30分钟内识别低至500 nM茶碱（图3）-暴露于靶标的样品制备物在步骤3中产生蓝色，而?…
Discussion
这里介绍的方法利用了变构核酶中最初无序的二级结构与通过靶标与适体结构域结合以激活顺式裂解锤头核酶而赋予的额外稳定性之间的过渡。调整核酶的稳定性，以在无靶标的情况下最小化催化活性，同时允许靶标结合恢复活性结构。此外，必须注意平衡促进EXPAR和显色所需的多种酶的缓冲液。最后，考虑到这是一个三级系统，孵育时间和温度以及反应组分浓度的组合意味着在最大化信号的同?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了Aptagen LLC的研发基金的支持。
Materials
| 3,3',5,5'-Tetramethylbenzidine dihydrochloride (TMB) solution with H2O2, "MaxSignal TMB Microwell Substrate Solution" | PerkinElmer | FOOD-1806-1000 | Color development reagent. Minimize exposure to light and atmosphere. Previously BIOO Scientific catalog number 1806. Supplied in Apta-beacon Demonstration Kit as Tube 3-2. |
| 5’- TCC CTC CCT CCC TCC CAG TCC AGA CTC TTC CCT CCC TCC CTC CCA GA-Biotin-3’ | Integrated DNA Technologies (IDT) | Custom | Optimized QtQ47 DNA template for EXPAR to produce G-quadruplex, primed by G-quadruplex. This is used for the "exponential" part of EXPAR, to rapidly increase the amount of DNAzyme used for Step 3 color development. Template includes a 3'-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. Part of Apta-beacon Demonstration Kit Tube 2-2. |
| 5’-GGG AAC UAU ACA ACC UAG GGC GAC CCU GAU GAG CCU UAU ACC AGC CGA AAG GCC CUU GGC AGA CGU UGA AAC GGU GAA AGC CGU AGG UUG CCC UAG GUU GUA UAG UU-3’ | Integrated DNA Technologies (IDT) | Custom | Theophylline-recognizing allosteric ribozyme sequence (self-cleaving ribozyme regulated by RNA aptamer domain for theophylline) modified from Soukup et al. Soukup, G. A., Emilsson, G. A., and Breaker, R. R. (2000) Altering molecular recognition of RNA aptamers by allosteric selection, Journal of molecular biology 298, 623-632. Supplied in Apta-beacon Demonstration Kit as Tube 1-3. |
| 5’-TCC CTC CCT CCC TCC CAG TCC AGA CTC TAC GGC TTT CAC CGT TTC AAC G-Biotin-3’ | Integrated DNA Technologies (IDT) | Custom | Optimized P3tQ49 DNA template for EXPAR to produce G-quadruplex, primed by P3 cleavage product. This is used in Step 2 to translate the cleavage product into DNAzyme used for Step 3 color development. Template includes a 3'-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. Part of Apta-beacon Demonstration Kit Tube 2-2. |
| 5x Ribozyme Buffer | Aptagen, LLC | N/A | 5x composition: 600 mM Tris-HCl (pH 7.5), 150 mM MgCl2, 25 mM DTT. Used in Step 1. Supplied in Apta-beacon Demonstration Kit as Tube 1-4. |
| Apta-beaconTM (GQ-EXPAR, TMB) Demonstration Kit | Aptagen, LLC | GQ-EXPAR-TMB | The demo kit showcases the specificity of the colorimetric assay by detecting difficult small molecule targets, theophylline versus caffeine, which only differ by a single methyl group. |
| Bst 2.0 DNA polymerase | New England Biolabs | M0537S | Isothermal amplification polymerase with strand-displacement activity. Part of the Nickase-Polymerase Mix, prepared at 9.375 U/uL. Part of Apta-beacon Demonstration Kit Tube 2-1. |
| Buffer 3.1 | New England Biolabs | B6003SVIAL | Combined with 2 uL of 10X Isothermal Amplification Buffer and 27.5 uL of nuclease-free water to produce 1.11X EXPAR Buffer in EXPAR Mix. This product replaces the previously-used B7203SVIAL that was part of the initial system development (same composition except non-recombinant BSA). Part of Apta-beacon Demonstration Kit Tube 2-2. |
| Caffeine | Sigma-Aldrich | C0750-100G | Aptamer counter-target (control), prepared with nuclease-free water. Supplied in Apta-beacon Demonstration Kit as Tube 1-1. |
| dNTPs | New England Biolabs | N0447S | Part of the EXPAR reaction mixture. Part of Apta-beacon Demonstration Kit Tube 2-2. |
| EXPAR reaction mixture | Aptagen, LLC | N/A | 0.44 mM dNTPs, 0.38 μM P3tQ49, 0.38 μM QtQ47, 44.44 mM Tris-HCl (pH 8.4), 63.5 mM NaCl, 31.5 mM KCl, 6.35 mM MgCl2, 1.27 mM MgSO4, 6.35 mM (NH4)2SO4, 63.5 μg/ml BSA, 0.0635 % Tween 20. Part of Apta-beacon Demonstration Kit Tube 2-2. |
| Hemin | Sigma-Aldrich | H9039 | Resuspended in DMF to a final concentration of 25 uM. Supplied in Apta-beacon Demonstration Kit as Tube 3-1. |
| Isothermal Amplification Buffer | New England Biolabs | B0537SVIAL | Combined with 2 uL of 10x Buffer 3.1 and 27.5 µL of nuclease-free water to produce 1.11x EXPAR Buffer in EXPAR Mix. Part of Apta-beacon Demonstration Kit Tube 2-2. |
| MgCl2 | Amresco (VWR) | E525-500ML | Hammerhead ribozyme cofactor, necessary for self-cleavage. Part of Apta-beacon Demonstration Kit Tube 1-3. |
| MJ PTC-100 Thermocycler | MJ Research, Inc. | PTC-100 | Thermocycler to control incubation temperatures. Any thermocycler or hot block can be used. |
| N, N-Dimethylformamide (DMF) | Sigma-Aldrich | 227056-100ML | Used to resuspend hemin and maximize shelf life in freezer. Part of Apta-beacon Demonstration Kit Tube 3-1. |
| Nickase-polymerase Mix | Aptagen, LLC | N/A | Nt.BstNBI (9.375 units/μL) and Bst 2.0 DNA polymerase (0.5 units/μL). Supplied in Apta-beacon Demonstration Kit as Tube 2-1. |
| Nt.BstNBI | New England Biolabs | R0607S | Nicking enzyme to allow continued isothermal amplification. Part of the Nickase-Polymerase Mix, prepared at 0.5 U/uL. Part of Apta-beacon Demonstration Kit Tube 2-1. |
| T4 Kinase Buffer | New England Biolabs | B0201SVIAL | Buffer for enzyme necessary to remove cyclic phosphate. Part of Apta-beacon Demonstration Kit Tube 1-4. |
| T4 polynucleotide kinase | New England Biolabs | M0201S | Removes cyclic phosphate post-cleavage to allow cleavage product to prime isothermal amplification reaction. Supplied in Apta-beacon Demonstration Kit as PCR Tubes. |
| Tecan GENios FL | Tecan | Genios-FL TWT | Plate reader to measure absorbance signal from Step 3 results. |
| Theophylline | Sigma-Aldrich | T1633-50G | Aptamer target, prepared with nuclease-free water. Supplied in Apta-beacon Demonstration Kit as Tube 1-2. |
| Tris-HCl | American Bioanalytical | AB14043-01000 | Aptamer binding buffer. Part of Apta-beacon Demonstration Kit Tube 1-4. |
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Cite This Article
Liao, A. M., Thoa, T. T. T., Caltagirone, G. T. Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction. J. Vis. Exp. (188), e64342, doi:10.3791/64342 (2022).