Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.

Recent studies have reported that mast cells play a role in the trafficking of dendritic cells and lymphocytes to lymph nodes draining sites of infection. To test the hypothesis that mast cell activators could be used as vaccine adjuvants, C3H/HeN mice were intradermally vaccinated in the ear on days 0 & 21 with 0.5 μg recombinant anthrax protective antigen (rPA) alone or combined with 30 μg compound 48/80 (C 48/80), a mast cell activator. CpG oligodeoxynucleotide (CpG ODN, 1 μg) was used as a control. Ear swelling was monitored 24 hours after immunization to measure injection site inflammation. Mice immunized with rPA alone had an average ear thickness (mm) of 0.0059 while mice immunized with rPA + C 48/80 had an ear thickness of 0.0426 (p < 0.001 vs rPA alone). Mice immunized with rPA + CpG ODN had an ear thickness of 0.1519 (p < 0.001 vs rPA), significantly greater than swelling induced by C 48/80 (p < 0.001). Day 42 serum from mice immunized with PA alone had serum anti-PA IgG titers of 1:27,238 while mice immunized with PA + C 48/80 had anti-PA titers of 1:10,568,984 (p < .001 vs PA alone). Similar titers were detected in mice vaccinated with rPA plus CpG ODN. Antibodies induced with rPA + C 48/80 neutralized anthrax lethal toxin. Our findings suggest that mast cell activators provide effective vaccine adjuvant activity when delivered intradermally while inducing less injection site inflammation than CpG ODN.