PNAs as Diagnostic Molecules in Malaria
2Department of Microbiology & Molecular Genetics, Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel
One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. The malaria parasite Plasmodium falciparum, which is the deadliest of all 5 types of Plasmodium parasites in humans, has been shown to gain resistance to key drugs such as artemisinin and chloroquine. The molecular mechanism of this resistance was found to be associated with single nucleotide polymorphisms (SNPs) in the K13 and CRT genes, respectively1,2.
PNAs (peptide nucleic acid) are synthetic oligonucleotides that can be used for diagnostic purposes by conjugation to a fluorophore. Interrupting the PNA sequence by a base surrogate bearing a “light up” fluorophore produces a FIT-PNA probe (forced intercalation) that fluoresces upon hybridization to the complementary DNA/RNA target.
We have designed and synthesized FIT-PNAs bearing the BisQ fluorophore that targets such an SNP in the K13 gene of P. falciparum. We demonstrate that these FIT-PNAs discriminate between WT and C580Y Plasmodium falciparum strains by observing a substantial difference in FIT-PNA fluorescence as corroborated by FACS analysis and confocal microscopy. We are now designing FIT-PNAs that target the K76T SNP associated with chloroquine resistance in P. falciparum. Providing a general tool to detect any SNP in P. falciparum may pave the way to develop a simple and sensitive assay that will provide the assessment for the optimal treatment for malaria-infected patients.
1 Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50-55 (2014).
2 Sidhu, A. B. S., Verdier-Pinard, D. & Fidock, D. A. Chloroquine Resistance in Plasmodium falciparum Malaria Parasites Conferred by pfcrt Mutations. Science 298, 210-213, doi:10.1126/science.1074045 (2002).
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