| The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7 and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen CG to TA base pairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT) 2 triplex to the corresponding Watson-Crick duplex and the T4(CTT) 2 overhang. The mass spectrometric analyses of (TTC)6·(GAA) 6o(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were only detectable at neutral and alkaline pH values. Mass spectrometric analysis also showed that minor groove binding ligands, berenil, netropsin and distamycin and the intercalating ligand, acridine orange, destabilize the (TTC)6·(GAA) 6o(TTC)6 triplex. These NMR and mass spectrometric methods might function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes associated with Friedreich's ataxia.; A mass spectrometry based assay was developed to screen inhibitors of beta amyloid protein aggregation. Alzheimer's disease (AD) is the most common cause of progressive decline of cognitive function in aged humans. Factors that facilitate beta amyloid protein aggregation are known to be one of the risk factors for AD. If compounds were discovered that could inhibit beta amyloid protein aggregation, then a treatment might be possible for AD. Sample preparation includes mixing beta amyloid protein (Abeta) with test compound, incubating the solution at 37°C for 20 hr, ultrafiltrating the mixture. Aliquots of the filtrate were analyzed using positive ion electrospray mass spectrometry. Quadruply charged protonated molecule of Abeta was detected at m/z 1083. Calibration curve was generated with correlation coefficient (r2) > 0.99 which indicates a linear detector response. The limit of detection was 0.224 ng (5.175 nM, 10 muL injection) and the limit of quantitation was 0.747 ng (17.25 nM, 10 muL injection). Melatonin, methysticin, 3-indolepropionic acid and daunomycin were chosen to demonstrate our assay and the order of inhibition activity is daunomycin > 3-indolepropionic acid > melatonin > methysticin. Finally, nine novel synthetic compounds (6-MeO-BTA-1-3-I, 6-Br-BTA-I, 6-HO-BTA-0-3'-I, 6-MeO-BTEP-1(E), 6-NH 2-BTP-1, 6-FEtO-BTA-0, 6-OH-BTA-1, 6-CN-BTA-1, BTA-N-PrF) where screened by using our assay for anti-aggregatory activity.; Finally, preliminary results of the application of the new mass spectrometry-based assay for screening of inhibitors of HbS aggregation, tubulin aggregation and potential application for screening of inhibitors of prion protein aggregation and PolyQ aggregation were discussed. |
