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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

Abstract

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

Introduction

Virus infections have enormous economic and societal impacts around the world, as became increasingly apparent from the recent COVID-19 pandemic. Timely and accurate diagnosis is paramount in treating viral infections while preventing the spread of viruses to healthy people. While many virus detection methods have been developed, such as PCR tests1,2 and inmunoassays3, most of the currently used methods are not capable of determining whether the detected virus is actually infectious or not. This is because the presence of components of the virus alone, such as viral nucleic acid or prot....

Protocol

1. Preparation of reagents and buffers

  1. Prepare 10x Tris-borate EDTA (10x TBE) by adding 0.9 M Tris-base, 0.9 M boric acid, 20 mM EDTA (disodium salt), and deionized water to a final volume of 1 L. Mix until all the components are dissolved.
  2. Prepare a 10% denaturing polyacrylamide stock solution as follows. In a 250 mL glass bottle, add 120 g of urea (8 M), 25 mL of 10x TBE, 62.5 mL of 40% acrylamide/bisacrylamide (29:1) solution, and enough distilled water to reach the final volume .......

Representative Results

Since DNA aptamers can be obtained using SELEX in a test tube15, this SELEX strategy was carefully designed to include both positive selection steps toward the intact, whole infectious virus (i.e., retain the DNA molecules that bind to the infectious virus), as well as counter selection steps for the same virus that has been rendered non-infectious by a particular disinfection method, specifically UV-treatment, by discarding the DNA sequences that can bind to the non-infectious virus. A schematic .......

Discussion

SELEX allows not only the identification of aptamers with high affinity, in the pM-nM range22,43,44,45, but also with high and tunable selectivity. By taking advantage of counter selection, aptamers with challenging selectivity can be obtained. For instance, the Li group has demonstrated the ability to obtain sequences that can differentiate pathogenic bacterial strains from non-pathogenic stra.......

Acknowledgements

We wish to thank Ms. Laura M. Cooper and Dr. Lijun Rong from the University of Illinois at Chicago for providing the pseudovirus samples used in this protocol (SARS-CoV-2, SARS-CoV-1, H5N1), as well as Dr. Alvaro Hernandez and Dr. Chris Wright of the DNA Services facility of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign for their assistance with high-throughput sequencing, and many members of the Lu group who have helped us with in vitro selection and aptamer characterization techniques. This work was supported by a RAPID grant from the National Science Foundation (CBET 20-29215) and a seed grant from the Institute f....

Materials

NameCompanyCatalog NumberComments
10% Ammonium persulfate (APS)BioRad1610700
100% EthanolSigma-AldrichE7023
1x PBS without calcium & magnesiumCorning21-040-CM
40% acrylamide/bisacrylamide (29:1) solutionBioRad1610146
Agencourt AMPure XP BeadsBeckman CoulterA63880DNA clean-up beads - Section 7.2.2
Amicon Ultra-0.5 Centrifugal Filter UnitMerckUFC501024cut-off 10 kDa
Amicon Ultra-0.5 Centrifugal Filter UnitMerckUFC510024cut-off 100 kDa
Boric AcidSigma-Aldrich100165
C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction ModuleBioRad1851148
Calcium ChlorideSigma-AldrichC4901
CFX Connect Real-Time PCR Detection SystemBioRad1855201
Digital Dry Baths/Block HeatersThermo Scientific88870001
Dynabeads MyOne Streptavidin C1Thermo Fisher65001streptavidin-modified magnetic beads - Section 4.9
EDTA disodium saltSigma-Aldrich324503
Eppendorf Safe-Lock microcentrifuge tubesSigma-AldrichT96611.5 mL
Lenti-X p24 Rapid Titer KitTakara Bio USA, Inc.632200Lentivirus quantification kit - Section 3.3.2.1
MagJET Separation Rack, 12 x 1.5 mL tubeThermo ScientificMR02
Magnesium chlorideSigma-AldrichM8266
Microseal 'B' PCR Plate Sealing Film, adhesive, opticalBioRadMSB1001non-UV absorbing
Mini-PROTEAN Tetra Cell for Ready Gel Precast GelsBioRad1658004EDU
Mini-PROTEAN Short PlatesBioRad1653308
Mini-PROTEAN Spacer Plates with 0.75 mm Integrated SpacersBioRad1653310
Molecular Biology Grade WaterLonza51200
Multiplate 96-Well PCR Plates, high profile, unskirted, clearBioRadMLP9611
Nanodrop OneThermo ScientificND-ONE-W
OneTaq DNA PolymeraseNew England BioLabM0480S
Ovation Ultralow v2 + UDITecan0344NB-A01High-troughput sequencing library preparation kit - Section 7.2.
PIPETMAN G (100-1000 µL, 20-200 µL, 2-20 µL and 0.2-2 µL)GilsonF144059M, F144058M, F144056M, F144054M
Purifier Logic+ Class II, Type A2 Biosafety CabinetsLabconco4261
Qubit dsDNA BR Assay KitInvitrogenQ32850fluorescence-based  dsDNA quantification  kit - Section 7.2.3
SHARP Classic Low Retention Pipet Tips (10 uL, 200 uL, 1000 uL)Thomas Scientific1158U43, 1159M44, 1158U40
Sodium acetateSigma-AldrichS2889
Sodium chlorideSigma-AldrichS7653
Sorvall Legend Micro 17R MicrocentrifugeThermo Scientific75002440
SsoFast EvaGreen SupermixBioRad1725201qPCR mastermix - Section 6.2.
Tris(hydroxymethyl)aminomethaneSigma-AldrichT1503
Tubes and Ultra Clear Caps, strips of 8USA scientificAB1183PCR tubes
UreaSigma-AldrichU5128

References

  1. Carter, L. J., et al. Assay techniques and test development for COVID-19 diagnosis. ACS Central Science. 6 (5), 591-605 (2020).
  2. Ranoa, D. R. E., et al. Saliva-based molecular testing for SARS-CoV-2 that bypasses RNA e....

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