FIT-PNA probes detect a point mutation associated with artemisinin resistance in malaria

PNAs (peptide nucleic acid) are synthetic DNA analogs that have been used for therapeutic (antisense) and diagnostic purposes. One diagnostic approach relies on the introduction of a cyanine dye replacing a natural base in the PNA sequence (FIT-PNA: forced intercalation-PNA). Such a surrogate base, when introduced into FIT-PNA, fluoresces only upon FIT-PNA hybridization to the complementary target DNA/RNA.

Plasmodium falciparum is the deadliest of human Plasmodium species, and has shown to gain resistance to the key antimalaria drug - artemisinin. This resistance is associated with single nucleotide polymorphisms (SNPs) in the K13 gene (Kelch-propeller protein).

We have designed and synthesized FIT-PNAs, bearing the BisQ cyanine dye as a surrogate base, that target the C580Y SNP in the K13 gene of P. falciparum. This SNP is a common SNP associated with artemisinin resistance. We have demonstrated that these FIT-PNAs discriminate between artemisinin-senritive strain (F2) and artemisinine-resistant strain (L18) of P. falciparum parasites.

In this work we coupled our FIT-PNA probes to Biotin and used Streptavidin-coated plates as a simple method for this SNP detection.

One major concern in using FIT-PNAs for SNP detection is the signal generated from the wild-type RNA transcript. Herein we present the use of small PNA molecules as competitive PNA molecules, targeting the WT strain, in order to reduce the WT fluorescence and improve SNP discrimination between SNP vs. WT synthetic RNAs.

We forsee FIT-PNAs as molecular probes that will provide fast, simple, and cheap means for the assessment of artemisinin resistance in malaria; a tool that would be highly desirable for the optimal choice of anti-malaria treatment in endemic countries.