Biomedical subjects
Publications and source records attributed to Robert F Bulleit.
New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.
Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease substrate and a firefly luciferase that has been molecularly evolved for increased stability. By combining the proluminescent caspase-3 substrate, Z-DEVD-aminoluciferin, with a stabilized luciferase in a homogeneous format, the authors developed an assay that is significantly faster and more sensitive than fluorescent caspase-3 assays. The assay has a single-step format, in which protease cleavage of the substrate and luciferase oxidation of the aminoluciferin occurs simultaneously. Because these processes are coupled, they rapidly achieve steady state to maintain stable luminescence for several hours. Maximum sensitivity is attained when this steady state occurs; consequently, this coupled-enzyme system results in a very rapid assay. The homogeneous format inherently removes trace contamination by free aminoluciferin, resulting in extremely low background and yielding exceptionally high signal-to-noise ratios and excellent Z' factors. Another advantage of a luminescent format is that it avoids problems of cell autofluorescence or fluorescence interference that can be associated with synthetic chemical and natural product libraries. This bioluminescent, homogeneous format should be widely applicable to other protease assays.
Studies of telomere length have been carried out in diverse areas of research. However, current methods to measure telomeres are cumbersome and not amenable to high-throughput analyses. Using a coupled pyrophosphorolysis/trans-phosphorylation reaction, we have developed a novel assay to quantitate telomere sequence content in a single tube or 96-well format. The method uses a telomere-specific oligonucleotide probe to sample nanogram quantities of DNA without PCR amplification. Polymerase and kinase enzymes drive the production of ATP, which is then monitored with a luciferase enzyme reporter system. Using this approach, we demonstrated that the luminescent output was linear across a 100-fold range of DNA input, and the assay was sensitive to 0.4-1 ng DNA. A control probe reaction and a DNA quantitation reaction were also designed using the same pyrophosphorolysis technology to correct for background activity and normalize the signal against variations in DNA input, respectively. Finally, we show that the normalized luminescent signal generated by this new method is highly correlated to the telomere restriction fragment length for six human cell lines.