Aptamer-mediated Norovirus Pull-Down

One of the objectives of this thesis was the development and discovery of a NoV aptamer to be used as an extraction tool. Therefore, the application of NoV aptamers in an aptamer-mediated pull-down assay to extract NoV from foods was investigated. Although previous studies using the FRA method showed that the aptamer-target binding might be impaired by the presence of food matrices, it was also discussed that the FRA method was not the ideal method to make this determination (see section 4.3). Therefore, all selected aptamers were tested in a pull-down assay (see section 3.8). The initial pull-down showed that the paramagnetic beads without aptamers (just beads=JB), which were used as a negative control in the virus pull-down, recovered virus from stool suspension. To be able to assess the pull-down abilities of the different aptamers, the beads needed to be blocked. Consequently, BSA blocking of the beads was implemented in the pull-down protocol, leading to a significant reduction (up to 1000-fold) of non-specific virus pull-pull-down of the beads without aptamers. Using the BSA blocking method, a NoV pull-down was tested with aptamers SMV 19, SMV 21, M 1, M 6-2, AG3, Beier, and Buf-2. The virus recoveries were compared to the negative control, by multiple comparison test. P-values were calculated indicating significant differences compared to the negative control. The pull-down was completed using partially purified stool suspension, containing GII.4 genotype Den Haag in two dilutions (100-fold and 1000-(100-fold).

For both dilutions, significantly more virus was recovered by aptamer-mediated pull-down, using aptamers SMV 19, SMV 21, M 1, M 6-2, Beier, and Buf-2, compared to the blocked paramagnetic beads. AG3 showed no significant difference to the negative control, of just the paramagnetic streptavidin beads. Results of the aptamer-mediated pull-down from the 100-fold dilution showed best virus recoveries, using aptamers M 1, and M 6-2, and SMV 21, reflected in the P-values of

<0,0001 compared to the negative control. SMV 19, Beier and Buf-2 mediated pull-downs completed from the 100-fold stool dilution, recovered less virus compared to aptamers M 1, M 6-2 and SMV 6-21, reflected in higher Ct values and a higher P-value of 0.006-21. Completing the pull-down from a 1000-fold stool dilution, no virus was recovered using the negative control and aptamer-mediated pull-down with AG3. The remaining aptamers tested, showed significantly higher recovery then the negative control. Overall, a similar NoV recovery pattern as observed for the 100-fold stool dilution was observed, completing pull-down s from a 1000-fold stool dilution.

Aptamer-mediated pull-downs using M 6-2, M 1, and SMV 21 showed the highest virus recovery, in descending order. The good results accomplished using aptamers M 1 and M 6-2 were not expected, as these aptamers did not show good binding characteristic in the FRA and dot-blot

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methods. The pull-down studies were completed using a different NoV GII.4 Den Haag (based on availability), a different genotype than used during the FRA binding studies. All aptamers showed genotype dependent differences in VLP binding, during FRA and dot-blot studies. Therefore, results observed during the pull-down differing from the previous studies could be due to genotype-specific binding of the aptamers. Hence, aptamers M 1, M 6-2, and SMV 19 could exhibit good binding characteristics for NoV GII.4 Den Haag.

Using the aptamers Beier, Buf-2 and M 1, M 6-2, SMV 19 and SMV 21, a NoV pull-down was completed from an artificially contaminated oyster sample and data from the pull-down compared to the existing FDA-shellfish method for NoV extraction from oysters. Results demonstrated the FDA-shellfish method had over 1000-fold better recovery than the best aptamer pull-down, accomplished with the SMV 21 aptamer. More importantly, it was shown that the blocking protocol developed using the BSA in every step of the pull-down, did not show the same efficiency in presence of the oyster matrix compared to the efficiency in buffer. The true potential of the aptamer-mediated pull-down can therefore not certainly be estimated.

Non-specific binding of viruses to paramagnetic beads are not a single occurrence. In a 2008 study an immunomagnetic approach to pull-down virus using antibodies was described, determining that monoclonal antibodies accomplished better NoV recovery then polyclonal antibodies²²⁸. However, a negative control employing beads without antibodies was not conducted. In a previous study investigating immunomagnetic pull-downs of enteric viruses it was noted that non-specific binding of viruses to paramagnetic beads was observed, but did not impair the limit of detection of the assay²²⁹. This makes sense for assays which follow the pull-down step with a subsequent specific detection step, like RT-qPCR, as commonly used for enteric RNA viruses.

However, the blocking of the virus’s non-specific binding to paramagnetic beads was imperative to determining the specificity of aptamer-mediated virus pull-downs. Additionally, it is noteworthy that the non-specific binding to paramagnetic beads is most probably not limited to only enteric viruses. Non-specific binding in pull-down assays could lead to the co-extraction of sample contaminants and PCR inhibitors, contradicting the idea of using a pull-down assay as purifying method in the first place.

M 1, and M 6-2 aptamers were previously studied in a pull-down application using BSA blocked paramagnetic streptavidin beads¹⁸⁹. However, in this study the negative control, using just the streptavidin beads was not prepared in skim milk, which was used as a blocking agent for aptamer-mediated pull-downs. Instead, the paramagnetic beads for the negative control were blocked with a commercial solution (Superblock T20, by Thermo Fisher Scientific). The results showed

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significantly better recoveries, compared to the negative control, during the pull-down for both aptamers in two of four stool dilutions tested. However, since the negative control did not represent the assay conditions, it is questionable whether these outcomes are truly a result of the aptamer pull-down or a result of non-specific binding to the skim milk blocked beads.

The results of the aptamer-mediated NoV pull-down from oysters suggest that the chemistry of the paramagnetic beads is not compatible with BSA blocking, which has also recently been noted by the manufacturer²³⁰. It is therefore necessary, to identify a suitable blocking agent before the aptamer-mediated pull-down can be further assessed. Additionally, the pull-down could be improved using an assortment of multiple NoV specific aptamers, increasing the cross reactivity and allow a more broad NoV detection, as suggested previously²³¹.

A further issue with the pull-down targeting the capsid protein, and using a genome-based detection method, is the fundamental occurrence of the virus and viral protein in clinical samples and the environment. It is well established for NoV and other enteric viruses that the presence of genomic viral RNA does not indicate whether the viral particle is intact⁷⁸; this is a problem as it suggests the presence of unassembled virus capsid in the sample. The pull-down depends on the retention of the entire viral particle on the paramagnetic bead to detect the capsid enclosed genome. The presence of unassembled capsid protein in stool samples would occupy aptamers and make them unavailable to bind an intact viral particle. Hence, potentially impairing the detection of NoV, as unassembled capsid protein is extracted but not identified by the detection method. This could be resolved by using an excess of aptamer and magnetic beads for the pull-down.

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