Preliminary development of DNA aptamer-Fc conjugate opsonins

Encapsulated bacteria such as virulent strains of Bacillus anthracis impair phagocytosis with their capsules unless opsonized by antibodies. Poly-gamma-D-glutamic acid (gamma-PDGA) is the major component of the B. anthracis capsule. In this work, poly-alpha-D-glutamic acid (alpha-PDGA)-coated magnetic beads (MBs) were used as surrogates to simulate vegetative B. anthracis cells and avoid the hazards of working with virulent bacteria. DNA aptamers were developed against the alpha-linked PDGA-MBs and sequenced. Four of the most frequent candidate aptamer sequences in the pool were coupled at their 5' ends to Fc fragments of murine IgG to act as artificial antibodies. The effects of candidate aptamer-Fc conjugate addition on macrophage attachment and internalization of alpha-PDGA-MBs were tested on P388D1 and RAW 264.7 murine macrophage lines by spectrofluorometric and image analysis techniques. P388D1 cells were not able to internalize the alpha-PDGA-MBs, but attachment to alpha-PDGA-MBs was enhanced by the conjugates to varying degrees. Ingestion of alpha-PDGA-MBs by RAW 264.7 cells in the presence of several different candidate aptamer-Fc conjugates demonstrated a statistically significant (p < 0.01) increase in phagocytic index (P.I.) up to threefold in the first 30 min of exposure to alpha-PDGA-MBs. This preliminary study using alpha-linked instead of gamma-linked PDGA provides proof-of-concept for future work in the new area of hybrid DNA aptamer-protein constructs as potential opsonins.