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

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.