Malaria, caused by protozoa of the genus Plasmodium, continues to be a life-threatening disease affecting nearly half the world’s population. Our ability to control and eradicate this disease has been hampered largely due to the spread of drug resistance. Further exacerbating this challenge is the parasite’s ability to evade drug pressure by entering a developmentally arrested state known as dormancy. P. falciparum, the deadliest of human infecting malaria parasites, enters dormancy as a response to artemisinin pressure, whereas P. vivax, the most widespread of malaria parasites, has naturally occurring dormant stages in the liver known as hypnozoites. The mechanisms behind both naturally occurring and drug-induced dormancy, and subsequent relapse or recrudescence, remain elusive and we lack the proper tools to study this phenomenon. While dormant cells do maintain low levels of metabolic activity, they do not actively synthesize DNA. Since Plasmodium is a purine auxotroph, we developed a novel tool using alkyne modified purines as DNA synthesis markers. We observed that modified adenosine can successfully differentiate P. vivax hypnozoites and liver stage schizonts and furthermore that it can capture reactivating hypnozoites. Additionally, modified adenosine, inosine, and hypoxanthine all incorporated into actively replicating P. falciparum, highlighting their utility for cell cycle studies. Alternatively, previous data has shown that exogenous addition of gibberellic acid to dihydroartemisinin-induced dormant P. falciparum stimulates early recrudescence. Therefore, we utilized gibberellic acid as a tool to investigate the mechanisms controlling dormancy and recrudescence. We modified gibberellic acid and localized it to the cytoplasm of all asexual blood stages of P. falciparum. Furthermore, we conducted pulldown studies and identified 50 proteins of interest involved in transcription, translation, post-translational modification, and protein trafficking. Among the proteins of interest was a putative AP2 transcription factor and several 26S proteasome subunit proteins. These classes of proteins have been associated with artemisinin resistance and could be involved in recrudescence from dihydroartemisinin-induced dormancy. Elucidating the parasite’s dormancy mechanisms is crucial towards malaria control and eradication, thus these studies provide the novel techniques necessary for future research of this phenomenon.
