1887

Abstract

Aptamers selected against various kinds of targets have shown remarkable specificity and affinity, similar to those displayed by antibodies to their antigens. To employ aptamers as genotyping reagents for the identification of pathogens and their strains, selections were carried out to find aptamers that specifically bind and distinguish the closely related human influenza A virus subtype H3N2. The selected aptamer, P30-10-16, binds specifically to the haemagglutinin (HA) region of the target strain A/Panama/2007/1999(H3N2) and failed to recognize other human influenza viruses, including another strain with the same subtype, H3N2. The aptamer displayed over 15-fold-higher affinity to the HA compared with the monoclonal antibody, and efficiently inhibited HA-mediated membrane fusion. These studies delineate the application of aptamers in the genotyping of viruses.

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2006-03-01
2019-11-14
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Inhibition of HA-mediated membrane fusion by P30-10-16C RNA. A time sequence of fluorescent images of virus–ghost conjugates during membrane fusion in the presence of complementary RNA P30-10-16C (5 µM) is demonstrated. After installing conjugates of R18-A/Panama and RBC ghosts on a fluorescence microscope at pH 7·5 and 20 ºC, a low-pH trigger (pH 5·0) was applied at time zero (the beginning of the video). The R18 fluorescence, which was originally constrained at punctate regions of individual virus particles, diffused out to the larger regions of the RBC ghosts, which indicates inter-membrane lipid mixing due to viral membrane fusion. The time sequence of 60 time-lapse images, recorded over 180 s (one frame every 3 s), is replayed with 36-fold-enhanced speed (12 frames s ). [ AVI file]

Inhibition of HA-mediated membrane fusion by P30-10-16 RNA. A time sequence of fluorescence images of virus–ghost conjugates after the low-pH trigger (pH 5·0 and 20 ºC, at time zero) in the presence of the selected aptamer P30-10-16 (5 µM) is demonstrated. In contrast to the result shown in Supplementary Video S1, R18 redistribution between the virus and the RBC ghost was not observed at most of the virus–ghost conjugates, indicating the inhibitory effect of the aptamer on viral membrane fusion. The images were recorded and replayed as described for Supplementary Video S1. [ AVI file]

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