Applications in Which Aptamers Are Needed or Wanted in Diagnostics and Therapeutics

Abstract

One strategy for bringing aptamers more into the mainstream of biomedical diagnostics and therapeutics is to exploit niche applications where aptamers are truly needed or wanted for their innate differences versus antibodies. This brief review article highlights some of those relatively rare applications in which aptamers are necessary or better suited to the user requirements than antibodies with explanations for why the aptamer is a necessary or superior choice. These situations include when no commercial antibody exists, when antibodies are excessively difficult to develop against a particular target because the target is highly toxic to host animals, when antibodies fail to discriminate closely related targets, when a smaller size is preferable to penetrate a tissue, when humanized monoclonal antibodies are too expensive and when the target is rapidly evolving or mutating. Examples of each are provided to illustrate these points.

Keywords: Cyclospora; SELEX; aptamer; diagnostic; humanized; liver fluke; monoclonal antibody; reproducibility; therapeutic; toxic.

Figure 1

Confocal fluorescence microscopy of Cyclospora cayetanensis oocysts after aptamer-based staining. Oocysts were stained with the top 8 DNA aptamer sequences containing 5′-biotin linkers at ~150 µg/mL in phosphate-buffered saline (PBS) for 30 min at room temperature and then washed by centrifugation at 13,000× g, and then the pelleted oocysts were resuspended in streptavidin–Texas Red conjugate for 15 min and washed again prior to confocal microscopy. Note that both the oocyst cell surface and interior structures (developing or developed spores) stained with each of the aptamers in panels (B–E) but not with a scrambled sequence DNA aptamer control shown in panel (A). Panel (F) shows the appearance of the unstained 8–10 µm oocysts under phase contrast microscopy. Total magnification = 400×.

Figure 2

Confocal fluorescence microscopy of anti-Cs44 aptamer-based fluorescence staining of adult Clonochis sinensis liver flukes in PBS using the final SELEX round 10 polyclonal aptamers and a method similar to that described in the Figure 1 legend, except that a fluoresceinated streptavidin conjugate was used to detect the 5′-biotinylated aptamer pool on the adult parasites’ surfaces in Panels (A,C). Panel (B) represents red autofluorescence of internal organs but no staining from an aptamer deletion control on the parasite’s surface. Panel (C) shows an image of combined fluorescence and brightfield confocal microscopy. Total magnification = 200×.

Figure 3

Confocal fluorescence microscopy of C. sinensis eggs stained by the same method described in Figure 2, except that the final 5′-biotinylated aptamer pool was raised against the recombinant C. sinensis egg protein. Panels (A,B) show egg surface staining, panel (C) represents the appearance of a negative aptamer deletion control and panel (D) illustrates the appearance of unstained eggs under phase-contrast microscopy. Total magnification = 400×.

Figure 4

Panel (A) illustrates the appearance of a Clonorchis Cs44 titration on lateral flow test strips using an aptamer–biotin–streptavidin–red Qdot 655 conjugate that migrated out of the conjugate pads near the bottom of the photograph to bind dried recombinant Cs44 protein lines at the levels indicated above the panel (i.e., 50 ng to 5 pg) with a detection limit of at least 5 pg versus the blank control as indicated by the capture lines shown at the arrow level. Panel (B) shows a similar preliminary capture test for 50 ng of the C. sinensis recombinant egg protein dried as a dot on the analytical membrane after interaction with an aptamer–biotin–streptavidin–Qdot 655 conjugate to provide proof of concept for eventual lateral flow test strips to detect liver flukes from human serum or diluted fecal suspensions.