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Bioengineering

体外选择核酸适配体以区分感染性和非感染性病毒

Published: September 07, 2022
doi:
10.3791/64127
, , ,
1INQUIMAE (CONICET), Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales,Universidad de Buenos Aires, 2Department of Chemistry,University of Illinois at Urbana-Champaign, 3Department of Chemistry,University of Texas at Austin

Summary

我们提供的方案通常可以应用于选择仅与感染性病毒结合的适配体，而不应用于通过消毒方法或任何其他类似病毒变得非传染性的病毒。这为在便携式和快速检测中确定感染性状态提供了可能性。

Abstract

病毒感染对社会有重大影响;大多数检测方法难以确定检测到的病毒是否具有传染性，导致治疗延误和病毒进一步传播。开发能够告知临床或环境样本可感染性的新型传感器将应对这一未满足的挑战。然而，很少有方法可以获得能够识别完整传染性病毒并将其与通过消毒方法变得非传染性的同一病毒区分开来的传感分子。在这里，我们描述了一种选择适配体的方案，该适配体可以通过指数富集（SELEX）对配体进行系统进化来区分感染性病毒和非感染性病毒。我们利用了SELEX的两个功能。首先，SELEX 可以量身定制，以使用计数器选择来去除竞争目标，例如非传染性病毒或其他类似病毒。此外，整个病毒可以用作SELEX的靶标，而不是例如病毒表面蛋白。全病毒SELEX允许选择与病毒天然状态特异性结合的适配体，而无需破坏病毒。因此，该方法允许基于病原体表面的功能差异获得识别剂，而不需要事先知道。

Introduction

病毒感染在世界各地具有巨大的经济和社会影响，最近的COVID-19大流行越来越明显。及时准确的诊断对于治疗病毒感染同时防止病毒传播给健康人至关重要。虽然已经开发了许多病毒检测方法，例如PCR测试1，2和免疫测定3，但目前使用的大多数方法都无法确定检测到的病毒是否真的具有传染性。这是因为仅存在病毒成分（例如病毒核酸或蛋白质）并不表明存在完整的传染性病毒，并且这些生物标志物的水平与感染性相关性较差4，5，6。例如，通常用于当前基于 PCR 的 COVID-19 测试的病毒 RNA 在感染的早期阶段患者具有传染性时水平非常低，而当患者从感染中康复并且不再具有传染性时，RNA 水平通常仍然很高7，8.病毒蛋白或抗原生物标志物遵循类似的趋势，但通常比病毒RNA出现得更晚，因此更不能预测感染性6，9。为了解决这一限制，已经开发了一些可以告知病毒感染状态的方法，但基于细胞培养微生物学技术，需要很长时间（几天或几周）才能获得结果4，10。因此，开发新的传感器，可以告知临床或环境样本的可感染性，可以避免治疗延误和病毒的进一步传播。然而，很少有方法可以获得能够识别完整感染性病毒粒子并将其与已变得非传染性的同一病毒区分开来的传感分子。

在这种情况下，核酸适配体特别适合作为独特的生物分子工具11，12，13，14。核酸适配体是具有特定核苷酸序列的短单链DNA或RNA分子，允许它们形成特定的3D构象以识别具有高亲和力和选择性的靶标15，16。它们是通过称为指数富集配体系统进化（SELEX）的组合选择过程获得的，也称为体外选择，该过程在具有10 14-1015序列的大型随机DNA采样文库的试管中进行 17，18，19.在该迭代过程的每一轮中，首先通过在所需条件下与靶标孵育来对DNA库进行选择压力。然后删除任何未与目标结合的序列，只留下那些在给定条件下能够结合的少数序列。最后，通过PCR扩增上一步中选择的序列，用下一轮选择所需的功能序列丰富池中的群体，并重复该过程。当选择池的活性达到平台期（通常在8-15轮后）时，通过DNA测序分析文库，以确定表现出最高亲和力的获胜序列。

SELEX具有独特的优势，可以利用这些优势来提高对其他类似靶标20，21的选择性，例如病毒22的感染性状态。首先，可以使用多种不同类型的靶标进行选择，从小分子和蛋白质到整个病原体和细胞16。因此，为了获得与感染性病毒结合的适配体，可以使用完整的病毒作为靶标，而不是病毒表面蛋白19。全病毒SELEX允许选择与病毒天然状态特异性结合的适配体，而无需破坏病毒。其次，SELEX可以定制以去除竞争靶标21，23，例如其他类似病毒或非传染性灭活病毒，使用每轮选择22中的计数器选择步骤。在计数器选择步骤中，DNA库暴露于不需要结合的靶标，并且任何结合的序列都被丢弃。

在这项工作中，我们提供了一种协议，该协议通常可用于选择与感染性病毒结合的适配体，但不能与通过特定消毒方法或另一种相关病毒结合的同一病毒结合。该方法允许基于病毒表面的功能差异获得识别剂，不需要事先知道，因此为检测新出现的病原体或研究不足的疾病提供了额外的优势。

Protocol

1. 试剂和缓冲液的制备 通过加入 0.9 M 三硼碱、0.9 M 硼酸、20 mM EDTA（二钠盐）和去离子水至最终体积为 1 L 来制备 10x 三硼酸盐 EDTA（10x TBE）。 制备10%变性聚丙烯酰胺储备溶液，如下所示。在 250 mL 玻璃瓶中，加入 120 g 尿素 （8 M）、25 mL 10x TBE、62.5 mL 40% 丙烯酰胺/双丙烯酰胺 （29：1） 溶液和足够的蒸馏水以达到 250 mL 的最终体积。混合直至所有成分溶解。 准备2…

Representative Results

由于DNA适配体可以在试管15中使用SELEX获得，因此该SELEX策略经过精心设计，既包括针对完整全传染性病毒的正向选择步骤（即保留与感染性病毒结合的DNA分子），也包括针对通过特定消毒方法变得无感染性的同一病毒的反选择步骤， 特别是紫外线处理，通过丢弃可以与非感染性病毒结合的DNA序列。选择过程的示意图如图 1所示。 作为?…

Discussion

SELEX不仅可以鉴定pM-nM范围为22，43，44，45的高亲和力适配体，还可以鉴定具有高可调选择性的适配体。通过利用计数器选择，可以获得具有具有挑战性选择性的适配体。例如，Li小组已经证明能够获得能够区分致病性细菌菌株和非致病菌株的序列21。此外，Le等人鉴定出一种能够区分<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢伊利诺伊大学芝加哥分校的Laura M. Cooper女士和Lijun Rong博士提供该协议中使用的假病毒样本（SARS-CoV-2，SARS-CoV-1，H5N1），以及伊利诺伊大学厄巴纳-香槟分校Roy J. Carver生物技术中心DNA服务设施的Alvaro Hernandez博士和Chris Wright博士在高通量测序方面的帮助， 以及Lu小组的许多成员，他们帮助我们进行 体外 选择和适配体表征技术。这项工作得到了美国国家科学基金会（CBET 20-29215）的RAPID资助以及伊利诺伊大学厄巴纳-香槟分校和伊利诺伊-JITRI研究所（JITRI 23965）的可持续、能源和环境研究所的种子资助。A.S.P. 感谢皮尤拉丁美洲奖学金的财政支持。我们还要感谢罗伯特·韦尔奇基金会（Grant F-0020）对德克萨斯大学奥斯汀分校Lu小组研究项目的支持。

Materials

10% Ammonium persulfate (APS) BioRad 1610700
100% Ethanol Sigma-Aldrich E7023
1x PBS without calcium & magnesium Corning 21-040-CM
40% acrylamide/bisacrylamide (29:1) solution BioRad 1610146
Agencourt AMPure XP Beads Beckman Coulter A63880 DNA clean-up beads – Section 7.2.2
Amicon Ultra-0.5 Centrifugal Filter Unit Merck UFC501024 cut-off 10 kDa
Amicon Ultra-0.5 Centrifugal Filter Unit Merck UFC510024 cut-off 100 kDa
Boric Acid Sigma-Aldrich 100165
C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction Module BioRad 1851148
Calcium Chloride Sigma-Aldrich C4901
CFX Connect Real-Time PCR Detection System BioRad 1855201
Digital Dry Baths/Block Heaters Thermo Scientific 88870001
Dynabeads MyOne Streptavidin C1 Thermo Fisher 65001 streptavidin-modified magnetic beads – Section 4.9
EDTA disodium salt Sigma-Aldrich 324503
Eppendorf Safe-Lock microcentrifuge tubes Sigma-Aldrich T9661 1.5 mL
Lenti-X p24 Rapid Titer Kit Takara Bio USA, Inc. 632200 Lentivirus quantification kit – Section 3.3.2.1
MagJET Separation Rack, 12 x 1.5 mL tube Thermo Scientific MR02
Magnesium chloride Sigma-Aldrich M8266
Microseal 'B' PCR Plate Sealing Film, adhesive, optical BioRad MSB1001 non-UV absorbing
Mini-PROTEAN Tetra Cell for Ready Gel Precast Gels BioRad 1658004EDU
Mini-PROTEAN Short Plates BioRad 1653308
Mini-PROTEAN Spacer Plates with 0.75 mm Integrated Spacers BioRad 1653310
Molecular Biology Grade Water Lonza 51200
Multiplate 96-Well PCR Plates, high profile, unskirted, clear BioRad MLP9611
Nanodrop One Thermo Scientific ND-ONE-W
OneTaq DNA Polymerase New England BioLab M0480S
Ovation Ultralow v2 + UDI Tecan 0344NB-A01 High-troughput sequencing library preparation kit – Section 7.2.
PIPETMAN G (100-1000 µL, 20-200 µL, 2-20 µL and 0.2-2 µL) Gilson F144059M, F144058M, F144056M, F144054M
Purifier Logic+ Class II, Type A2 Biosafety Cabinets Labconco 4261
Qubit dsDNA BR Assay Kit Invitrogen Q32850 fluorescence-based  dsDNA quantification  kit – Section 7.2.3
SHARP Classic Low Retention Pipet Tips (10 uL, 200 uL, 1000 uL) Thomas Scientific 1158U43, 1159M44, 1158U40
Sodium acetate Sigma-Aldrich S2889
Sodium chloride Sigma-Aldrich S7653
Sorvall Legend Micro 17R Microcentrifuge Thermo Scientific 75002440
SsoFast EvaGreen Supermix BioRad 1725201 qPCR mastermix – Section 6.2.
Tris(hydroxymethyl)aminomethane Sigma-Aldrich T1503
Tubes and Ultra Clear Caps, strips of 8 USA scientific AB1183 PCR tubes
Urea Sigma-Aldrich U5128
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Tags

Aptamer SelectionIn Vitro SelectionInfectious VirusNon-infectious VirusSELEXAffinity-based SelectionVirus DetectionVirus InfectivityDiagnostic SensingEnvironmental Monitoring
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Gramajo, M. E., Lake, R. J., Lu, Y., Peinetti, A. S. In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses. J. Vis. Exp. (187), e64127, doi:10.3791/64127 (2022).
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