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.

Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection.

DNA aptamers were developed against MgCl(2)-extracted surface proteins from Campylobacter jejuni. The two highest affinity aptamers were selected for use in a magnetic bead (MB) and red quantum dot (QD)-based sandwich assay scheme. The assay was evaluated using both heat-killed and live C. jejuni and exhibits detection limits as low as an average of 2.5 colony forming unit (cfu) equivalents in buffer and 10-250 cfu in various food matrices. The assay exhibits low cross-reactivity with bacterial species outside the Campylobacter genus, but exhibits substantial cross-reactivity with C. coli and C. lari. The assay was evaluated with a spectrofluorometer and a commercially available handheld fluorometer, which yielded comparable detection limits and ranges. Remarkably, the sandwich assay components adhere to the inside face of polystyrene cuvettes even in food matrices near neutral pH, thereby enabling a rapid homogeneous assay, because fluorescence is concentrated to a small, thin planar area and background fluorescence from the bulk solution is minimized. The plastic cuvette-adherent technology coupled to a sensitive handheld fluorometer may enable rapid (15-20 min), portable detection of foodborne pathogens from "farm-to-fork" by obviating the slow enrichment culture phase used by other food safety tests.

Selective glutaraldehyde-mediated coupling of proteins to the 3'-adenine terminus of polymerase chain reaction products.

Attachment of proteins to the 3' end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ' modifications by solid-phase synthesis for protein attachment is limited to 45-60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3'-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N6 position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4 degrees C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and beta2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4 degrees C showed no evidence of DNA-DNA or DNA-protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA-3'-protein conjugates might be easily mass-produced. The protein portion of a DNA-3'-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.