It is widely known that the majority of compounds used in high-throughput screening and aimed at preventing tau protein aggregation quench the thioflavin T (ThT) fluorescence. Using ThS/T (thioflavin S or T) for the monitoring of amyloid aggregation has additional disadvantages. For instance, the emission spectrum is largely dominated by ThS/T molecules attached to tau aggregates, whereas tau monomers, dimers, and oligomers are almost invisible. As a consequence, the ratio of soluble tau monomers to the aggregated protein is not possible to quantify using ThS/T. This leads to the difficulty in characterizing the initial stages of monomer→dimer→oligomer→aggregate process, which are essential for understanding the nature and mechanism of amyloid formation pathology. Finally, ThS/T molecule is an additional reaction component, which leads to two consequences: (i) the overall tau aggregation process more complex to analyze; and (ii) ThS/T might interfere with the small-molecule compounds binding to tau protein.
Heparin is known to induce strong and fast (several hours) tau aggregate formation. Working with heparin in any biochemical assay can be notoriously difficult due to two reasons: (i) the omnipotent heparin binding to virtually any protein surface or small-molecule having a positive charge almost irreversibly under native conditions; and (ii) the order-of-addition in vitro reactions require extensive optimization and careful heparin dose titration. It has to be noted, however, that the actual molecular mechanism for tau aggregate formation is not known. Clearly, heparin-induced tau aggregate formation is not the true pathological pathway.
In this Solution, NextGenRnD reports novel quantitative, robust, and high-throughput assay for monitoring tau protein aggregation. This in vitro assay is cell-free, heparin-free, and does not depend on ThS/T.