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 for Sustainability, Energy, and Environment at the University of Illinois at Urbana-Champaign and Illinois-JITRI Institute (JITRI 23965). A.S.P. thanks the PEW Latin American Fellowship for financial support. We also thank the Robert A. Welch Foundation (Grant F-0020) for support of the Lu group research program at the University of Texas at Austin.

1. Preparation of reagents and buffers
<ol>
	<li>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.</li>
	<li>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 ...

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...

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 proteins, does not indicate that the intact, infectious virus is present, and levels of these biomarkers have shown poor correlation with infectivity4,5,6. For example, viral RNA, commonly used for the current PCR-based COVID-19 tests, has very low levels in the early stages of infection when the patient is contagious, while the RNA level is often still very high when patients have recovered from the infection and are no longer contagious7,8. The viral protein or antigen biomarkers follow a similar trend, but typically appear even later than the viral RNA and thus are even less predictive of infectability6,9. To address this limitation, some methods that can inform on the infectivity status of the virus have been developed, but are based on cell culture microbiology techniques that require a long time (days or weeks) to obtain results4,10. Thus, developing new sensors that can inform on the infectability of clinical or environmental samples can avoid delays in treatment and further spread of the virus. However, very few methods can obtain sensing molecules that can recognize an intact infectious virion and differentiate it from the same virus that has been rendered non-infectious.
In this context, aptamers are particularly well-suited as a unique biomolecular tool11,12,13,14. Aptamers are short, single-stranded DNA or RNA molecules with a specific nucleotide sequence that allows them to form a specific 3D conformation to recognize a target with high affinity and selectivity15,16. They are obtained by a combinatorial selection process called systematic evolution of ligands by exponential enrichment (SELEX), also known as in vitro selection, that is carried out in test tubes with a large random DNA sampling library of 1014-1015 sequences17,18,19. In each round of this iterative process, the DNA pool is first subjected to a selection pressure through incubation with the target under the desired conditions. Any sequences that are not bound to the target are then removed, leaving behind only those few sequences that are able to bind under the given conditions. Finally, the sequences that have been selected in the previous step are amplified by PCR, enriching the population of the pool with the desired functional sequences for the next round of selection, and the process is repeated. When the activity of the selection pool reaches a plateau (typically after 8-15 rounds), the library is analyzed by DNA sequencing to identify the winning sequences exhibiting the highest affinity.
SELEX has unique advantages that can be exploited to gain increased selectivity against other similar targets20,21, such as for infectivity status of the virus22. First, a wide variety of different types of targets can be used for the selection, from small molecules and proteins to whole pathogens and cells16. Thus, to obtain an aptamer that binds to an infectious virus, an intact virus can be used as the target, instead of a viral surface protein19. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need for disruption of the virus. Second, SELEX can be tailor-made to remove competing targets21,23, such as other similar viruses or non-infectious inactivated viruses, using counter selection steps in each round of selection22. During the counter selection steps, the DNA pool is exposed to targets for which binding is not desired, and any sequences that bind are discarded.
In this work, we provide a protocol that can be generally applied for selecting aptamers that bind to an infectious virus but not to the same virus that has been rendered non-infectious by a particular disinfection method or to another related viruses. This method allows recognition agents to be obtained based on functional differences of the virus surface, which do not need to be known in advance, and so offers an additional advantage for the detection of newly emerged pathogens or for understudied diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Ammonium persulfate (APS)</td>
			<td>BioRad</td>
			<td>1610700</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS without calcium &#38; magnesium</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>40% acrylamide/bisacrylamide (29:1) solution</td>
			<td>BioRad</td>
			<td>1610146</td>
			<td></td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>DNA clean-up beads - Section 7.2.2</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Merck</td>
			<td>UFC501024</td>
			<td>cut-off 10 kDa</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Merck</td>
			<td>UFC510024</td>
			<td>cut-off 100 kDa</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>100165</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction Module</td>
			<td>BioRad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>BioRad</td>
			<td>1855201</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Dry Baths/Block Heaters</td>
			<td>Thermo Scientific</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>Thermo Fisher</td>
			<td>65001</td>
			<td>streptavidin-modified magnetic beads - Section 4.9</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>324503</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock microcentrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>T9661</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Lenti-X p24 Rapid Titer Kit</td>
			<td>Takara Bio USA, Inc.</td>
			<td>632200</td>
			<td>Lentivirus quantification kit - Section 3.3.2.1</td>
		</tr>
		<tr>
			<td>MagJET Separation Rack, 12 x 1.5 mL tube</td>
			<td>Thermo Scientific</td>
			<td>MR02</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>BioRad</td>
			<td>MSB1001</td>
			<td>non-UV absorbing</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell for Ready Gel Precast Gels</td>
			<td>BioRad</td>
			<td>1658004EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Short Plates</td>
			<td>BioRad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Spacer Plates with 0.75 mm Integrated Spacers</td>
			<td>BioRad</td>
			<td>1653310</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Lonza</td>
			<td>51200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplate 96-Well PCR Plates, high profile, unskirted, clear</td>
			<td>BioRad</td>
			<td>MLP9611</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Thermo Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>OneTaq DNA Polymerase</td>
			<td>New England BioLab</td>
			<td>M0480S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Ultralow v2 + UDI</td>
			<td>Tecan</td>
			<td>0344NB-A01</td>
			<td>High-troughput sequencing library preparation kit - Section 7.2.</td>
		</tr>
		<tr>
			<td>PIPETMAN G (100-1000 &#181;L, 20-200 &#181;L, 2-20 &#181;L and 0.2-2 &#181;L)</td>
			<td>Gilson</td>
			<td>F144059M, F144058M, F144056M, F144054M</td>
			<td></td>
		</tr>
		<tr>
			<td>Purifier Logic+ Class II, Type A2 Biosafety Cabinets</td>
			<td>Labconco</td>
			<td>4261</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td>fluorescence-based&#160; dsDNA quantification&#160; kit - Section 7.2.3</td>
		</tr>
		<tr>
			<td>SHARP Classic Low Retention Pipet Tips (10 uL, 200 uL, 1000 uL)</td>
			<td>Thomas Scientific</td>
			<td>1158U43, 1159M44, 1158U40</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 17R Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>1725201</td>
			<td>qPCR mastermix - Section 6.2.</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes and Ultra Clear Caps, strips of 8</td>
			<td>USA scientific</td>
			<td>AB1183</td>
			<td>PCR tubes</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro selection of aptamers to differentiate infectious from non-infectious viruses

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.

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 ...

Watch this Scientific Journal Video about In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses at JoVE.com

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

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.

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, ...

This protocol describes how to select DNA aptamers that can distinguish infectious viruses versus non-infectious viruses in order to obtain sensing molecule able to inform the infectability of clinical or environmental samples. This technique can be applied to obtain selective aptamers for many other viruses, including emerging viruses, because it does not require prior knowledge of the virus'structures or sequences. This technique allows the development of aptamer sensors for clinical diagnostic and environmental monitoring of infectious viruses to detect if someone is contagious or if an environmental disinfection method work as intended.Selection can be a difficult process, especially for those who are new to it. It's important to optimize the PCR conditions and to monitor the progress of the selection with qPCR. Demonstrating the procedure will be Marcos Gramajo, a graduate student from my lab.To begin, design the initial single-stranded or SS-DNA library and primers. Obtain the DNA oligos from commercial sources with standard desalting purification. Purify SS-DNA library and primers on 10%denaturing polyacrylamide gel electrophoresis followed by ethanol precipitation.Next, obtain a stock solution of infectious and non-infectious viruses or prepare them as described in the text manuscript. For denaturation of SS-DNA library, mix 10 microliters of 100 micromolar SS-DNA library with 240 microliters of SELEX buffer. Heat the mixture at 95 degrees Celsius for 15 minutes in a dry bath, then place the tube on ice for 15 minutes.Mix 250 microliters of SS-DNA library in SELEX buffer with 50 microliters of infectious virus for positive selection. Incubate for two hours at room temperature. Then add 400 microliters of one millimolar T20 sequence to a 0.5 milliliter filter to block the non-specific sites.Incubate the filters for 30 minutes. Centrifuge the filter at 14, 000 G for 10 minutes to remove the T20 solution. Then wash the filter thrice with SELEX buffer to remove excess T20 sequences.Next, add SS-DNA library infectious virus mixture to the blocked 100 kilodalton filter and centrifuge to wash unbound sequences. Wash the filter thrice by adding 400 microliters of SELEX buffer and centrifugation. Keep the fraction in the filter and discard the flow-through.To elute bound sequences, change the collection tube of the centrifuge filter and add 300 microliters of SELEX buffer containing eight molar urea to the filter. Heat the filter at 95 degrees Celsius for 15 minutes, then centrifuge to collect the flow-through containing the eluted sequences. Once done, wash the materials with 10%bleach.Next, add the SS-DNA infectious virus solution collected in the earlier step to a blocked 10 kilodalton filter, centrifuge at 14, 000 G for 15 minutes and discard the flow-through. Wash the filter thrice with 300 microliters of SELEX buffer to remove the urea by centrifugation and discard the flow-through. Recover the solution in the filter by turning it upside down in a clean collection tube and centrifuging for five minutes.Measure the final volume of the recovered solution using a pipette and label it as a Round 1A sample. Take 90%of the Round 1A sample as a template in the first round and run the PCR using optimized conditions. To recover SS-DNA using streptavidin modified magnetic beads or MB, split the product into 50 microliter aliquots and add them to microcentrifuge tubes containing 50 microliters MB.Incubate the tube for 30 minutes with mild agitation at room temperature.Then place the tube on the magnetic rack to isolate the MB and remove the supernatant by pipetting. Wash the MB twice by adding 200 microliters of binding and washing buffer to the tube. Then remove the tube from the magnetic rack and tap it before resuspending the MB to a homogenous solution by pipetting.Place the tube again on the magnetic rack and remove the supernatant. After the second wash, resuspend the MB in 100 microliters of SELEX buffer and heat the solution at 95 degrees Celsius for 10 minutes. Immediately place the tube in the magnetic rack and collect the supernatant containing the SS-DNA pool.Repeat this recovery step adding 50 microliters of SELEX buffer. Measure the final volume of the recovered fraction and label it as Round 1X. For denaturation of the SS-DNA pool, take 60%of the Round 1X sample and mix it with 50 microliters of SELEX buffer.Heat the sample in a dry bath, then place the tube on ice for 15 minutes. Next, mix the denatured SS-DNA pool in SELEX buffer with 50 microliters of each virus as the counter-selection step and incubate for one hour at room temperature. After blocking the non-specific sites in centrifuge filters, add the mixture denatured SS-DNA pool incubated with a virus solution to a blocked 100 kilodalton filter.Centrifuge the filter at 14, 000 G for 10 minutes and collect the flow-through. Take 300 microliters of fraction and mix with 50 microliters of infectious virus containing 100 million copies per milliliter. Incubate for two hours at room temperature.Prepare a standard qPCR master mix and dilutions of purified SS-DNA library as described in the text. Run the qPCR to determine the threshold cycle. After the run, use a standard curve to quantify the amount of SS-DNA in Round 1A and Round 1X.Then calculate the elution yield as the bound DNA amount divided by the initial DNA amount and plot the elution yield versus the round number. Finally, plot the melting curves for different rounds. For high throughput sequencing, select an appropriate commercially available kit.Prepare multiple pools of selection rounds representing high, medium, and low enrichment, as well as the final pool using a different pair of indexes in the adapters for each pool. Then send the prepared final library to the sequencing facility. For sequencing analysis, sequences have been de-multiplexed by the sequencing facility.Remove the sequencing adapters and selection primers using the command line program Cutadapt. Then use the FASTX-Toolkit program to remove low-quality sequences. Then merge the sequence files in the reverse direction with those in the forward direction using the FASTX-Toolkit FASTX_reverse_ complement function on the reverse direction files.Then use the built-in CAT program to merge the files into a single file. Use the FASTAptamer analysis toolkit to analyze the enrichment of sequences across different selection pools. First, to use the FASTAptamer count and FASTAptamer cluster functions to group similar sequences into clusters.Next, use the FASTAptamer enrich function to get information on the enrichment of sequences and clusters. Identify the candidate aptamer sequences from the enrichment information obtained through sequence analysis. Select several aptamer sequences and perform initial screening using a binding assay.In this analysis, qPCR was used to monitor the progress of SELEX of infectious SARS-CoV-2 specific aptamers. The elution yield initially increased with each round of SELEX and leveled at rounds eight and nine. By comparing the melting curves, a shift from the peak at a high melting temperature from 77 to 79 degrees Celsius was observed.Moreover, in the last two rounds, a peak at a low melting temperature was observed. The relative abundance in reads per million for the sequenced SARS2 AR10 obtained over consecutive selection rounds is shown. The predicted structure of SARS2 AR10 showed a structured secondary structure containing a stem loop region that might be involved in recognizing the virus.Once aptamers are obtained, they can be incorporated into electrical opticals, another type of sensors, to develop portal and rapid virus detection test that can inform virus infectivity. We envision that aptamer specific to different variants or stereotypes of virus can be obtained, making it possible for rapid monitoring of variants that are the biggest threat to society.

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Abstract