Materials:
… PA from B. anthracis in its holo form (PA83) and cleaved form (PA63) where purchased from List Biological Laboratories, …
In vitro selection scheme:
The AEGIS-LIVE experiment was performed on a synthetic library of GACTZP oligonucleotides containing 25 randomized positions flanked by two primer binding sites (59 nt in length, 5-AGAGAGCGTCGTGTGGA-N25-TGAGGAGGTGCGCAAGT-3).
PA was presented immobilized on magnetic beads…
Filter binding assays:
5-32P-labeled aptamer (1 l each) was denatured/renatured in 1 PBS (30 l), and incubated (45 min, RT) with increasing concentrations of PA63 or PA83, from 0 to 12 M. To capture the PA and PA-aptamer complex, aliquots (5 l) from each binding reaction were spotted on a nitrocellulose membrane on top of a charged nylon membrane (Whatman) in a dot-blot minifold vacuum device (Millipore) (27). Membranes were washed …
Determination of the affinity constant with bead-based assay:
After being coupled to PA63 (1.6 nmoles), the beads (200 l, as during selection) were divided into aliquots (20 l each) and each fraction was incubated with increasing concentrations of …
Enzymatic footprinting:
For DNase I digestion of PA1 and PA1T4 in the presence of PA63 or PA83, the aptamer was denatured (85C, 5 min) followed by slow (0.1/s) cooling to 24C to allow refolding. 532P-PA1 with 05 M PA63 or PA83 was incubated with tumbling for 45 min, and then treated with DNase I nuclease (15 min at RT). The digested products were resolved on 18% denaturing PAGE. …
Electrophysiology assays:
LF N-terminal domain (LFN) and PA were expressed and purified as described (21). To form heptameric PA oligomers, PA was nicked with trypsin (1:1000 w/w) at room temperature in buffer (20 mM TrisCl, pH 8) …