Use of pteridine nucleoside analogs as hybridization probes.

The pteridine nucleoside analog 3-methyl isoxanthopterin (3-MI) is highly fluorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligonucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its fluorescence is variably quenched in a sequence-dependent manner. Bend ing, annealing, binding, digestion or cleavage of fluorophore-containing oligonucleotides can be detected by monitoring changes in fluorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the fluorophore as an insertion and when annealing occurs, the fluorophore bulges out from the double strand, resulting in increased fluorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple fluorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybridization technique can result in fluorescence intensity increases of up to 27-fold. Fluorescence intensity increases are only seen upon specific binding to bulge-generating complements, removing issues of high background from non-specific binding.

Stimulus-dependent production of cytokines and pterins by peripheral blood mononuclear cells.

The cytokine profiles produced by peripheral blood mononuclear cell (PBMC) cultures were dependent upon the nature of the stimulus used. Powerful lymphocyte activators such as mitogens induced rapid cell proliferation together with the production of both inflammatory (IL-1 alpha, IL-1 beta, IL-6 and TNF alpha) and immune (IFN-gamma, TNF-alpha and TNF-beta) cytokines, and immune activation markers (soluble IL-2 receptor, neopterin and xanthopterin). Bacterial endotoxin failed to induce cell proliferation but resulted in the rapid production of inflammatory cytokines together with a short burst of IFN-gamma production, without the production of the other immune cytokines or activation markers. Alloantigen stimulation gave a typical immune cytokine and marker profile, with little or no production of inflammatory cytokines. Re-call antigens (candida and PPD) induced maximal cell proliferation at days 5 to 6, but induced little or no production of inflammatory cytokines. Markedly different immune cytokine profiles were obtained with these re-call antigens. Candida induced an early burst of IFN-gamma production on day 1 followed by later production of TNF-alpha. In cultures stimulated with PPD, both IFN-gamma and TNF-alpha were detected from day 2. With both re-call antigens, the levels of production of the activation markers were equivalent to the proliferative responses obtained.