In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine–dithiazole (QCy–DT) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCy–DT exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCy–DT for DNA containing 5′-AATT-3′ sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5′-X(AATT)Y-3′ recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC50 value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC50 make QCy–DT a potential and commercially viable DNA probe.
Even though it is increasingly evident that post-transcriptional events like mRNA processing and splicing may regulate gene expression and proteome diversity of malaria parasite Plasmodium, molecular mechanisms that regulate events like mRNA splicing in malaria parasite are poorly understood. Protein kinases control a wide variety of cellular events in almost all eukaryotes, including modulation of mRNA splicing, transport, and stability. We have identified a novel splicing-related protein kinase from Plasmodium falciparum, PfSRPK1. PfSRPK1 when incubated with parasite nuclear extracts inhibited RNA splicing, suggesting that it may control mRNA splicing in the parasite. PfSR1, a putative splicing factor from P. falciparum, was identified as a substrate of PfSRPK1. PfSR1 interacts with RNA and PfSRPK1 modulates its RNA binding. Early in the parasite development, PfSRPK1 and PfSR1 are present in the nucleus. These studies provide useful insights into the function of two potentially key components of P. falciparum mRNA splicing machinery.Malaria is one of the most serious infectious diseases and causes several million deaths and clinical illness in hundreds of millions of people every year (1). One of the major problems of recent times has been the emergence of new drugresistant strains of the malaria parasite Plasmodium falciparum. After invasion of the erythrocytes, the parasite can propagate asexually, giving rise to several new merozoites that invade fresh erythrocytes, which serve as hosts for the parasite to propagate. The blood stage infection is the cause of malaria pathogenesis. The parasite also undergoes sexual differentiation, which leads to the formation of male and female gametocytes. Upon ingestion of gametocytes by the Anopheles mosquito, further development continues inside the vector host. It is well known that signal transduction events mediated by protein kinases regulate diverse cellular processes. The importance of protein kinases in malaria parasite has been highlighted by a series of recent reports (2, 3), as these enzymes have been implicated in wide ranging events in both asexual and sexual stages of the parasite life cycle and, therefore, are considered as potential drug targets. Despite these reports, the information regarding signaling networks in the parasite is very limited.Although it is clear that metazoan protein kinase cascades control gene expression by regulating transcriptional or posttranscriptional events, this area in P. falciparum biology is largely unexplored. Plasmodium displays a high degree of developmental control of gene expression (4). Strikingly, only a few transcription factors have been identified in the parasite leading to the speculation that post-transcriptional events may be pivotal in regulating gene expression in the parasite (5). Translational repression of some genes (6) and control of gene expression by antisense RNA are some of the post-transcriptional (7) events that have been implicated in regulating gene expression in the parasite. mRNA splicing is on...
Calcium-dependent protein kinases (CDPKs) are major effectors of calcium signaling in apicomplexan parasites like Toxoplasma and Plasmodium and control important processes of the parasite life cycle. Despite recently reported crystal structures of Toxoplasma gondii (Tg)CDPKs, several important questions about their regulation remain unanswered. Plasmodium falciparum (Pf)CDPK1 has emerged as a key player in the life cycle of the malaria parasite, as it may be involved in the invasion of the host cells. Molecular modeling and site-directed mutagenesis studies on PfCDPK1 suggested that several residues in the regulatory domain play a dual role, as they seem to contribute to the stabilization of both the active and inactive kinase. Mass spectrometry revealed that PfCDPK1 was autophosphorylated at several sites; some of these were placed at strategic locations and therefore were found to be critical for kinase activation. The N-terminal extension of PfCDPK1 was found to be important for PfCDPK1 activation. Unexpectedly, an ATP binding site in the NTE of PfCDPK1 was identified. Our studies highlight several novel features of PfCDPK1 regulation, which may be shared by other members of the CDPK family. These findings may also aid design of inhibitors against these important targets, which are absent from the host.
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