Predictive Model for the Sequence-Dependent Fluorogenic Response of Forced-Intercalation Peptide Nucleic Acid

Abstract

The forced-intercalation peptide nucleic acid (FIT-PNA) concept, introduced by Seitz and co-workers, is based on replacing a nucleobase of the PNA sequence with a cyanine dye (such as thiazole orange). The cyanine dye is thus a surrogate base that is forced to intercalate in the duplex (e.g., PNA:DNA). This allows single-mismatch sensitivity as the introduction of a mismatch in the vicinity of the dye increases freedom of motion and leads to a significant depletion of its fluorescence because of the free rotation of the monomethine bond separating the two π-systems of the cyanine dye. Herein, we designed and synthesized six FIT-PNA probes, featuring bisquinoline (BisQ), a red-emitting cyanine dye recently developed in our laboratory for FIT-PNAs. By following PNA–DNA duplex fluorescence, we found new sequence-based factors governing the fluorescence response to the mismatched FIT-PNA:DNA duplex. Fluorogenic properties are correlated with the π-stacking energy of three distinctive base pair steps (BPSs) in the PNA:DNA duplex. The first two are the two BPSs opposite BisQ, whereas the third is the BPS of the mismatch position, which presumably becomes unstacked due to the mismatch. We suggest a predictive model for FIT-PNA single-mismatch detection mechanism, a model that can be used in future research to improve FIT-PNA design.

FIT-PNAs as RNA-Sensing Probes for Drug-Resistant Plasmodium falciparum

“…These features have prompted us to design FIT-PNAs (with an appended octa-D-lysine CPP) as a means for simple detection of artemisinin resistance in P. falciparum. Previously, 51 we have synthesized six FIT-PNA probes complementary to the mutant C580Y K13 mRNA. In this earlier report, we studied the fluorogenic properties of these FIT-PNAs with synthetic DNA.…”

“…In this earlier report, we studied the fluorogenic properties of these FIT-PNAs with synthetic DNA. 51 Herein, we examined the sensing properties of all six FIT-PNAs for the C580Y SNP and one newly synthesized FIT-PNA complementary to the mutant CRT mRNA in the Dd2 strain (Table 1 and Figures S5 and S6). In addition, we have prepared two FIT-PNAs (WT K13 FIT-PNAs 1 and 2) that target the wild-type sequence of the pf K13 gene (Table 1 and Figures S1−S4).…”

“…falciparum. Previously, we have synthesized six FIT-PNA probes complementary to the mutant C580Y K13 mRNA. In this earlier report, we studied the fluorogenic properties of these FIT-PNAs with synthetic DNA .…”

“…Synthesis of FIT-PNAs. Six K13 FIT-PNA probes (Table 1) were previously synthesized and fully characterized by HPLC and ESI-MS. 51 WT K13 FIT-PNAs (1 and 2), K67T FIT-PNA, and NT FIT-PNA were synthesized on the solid support (Novasyn TGA resin, 0.25 mmol/g) by standard peptide chemistry using Fmoc-protected PNA monomers. 52 HPLC and ESI-MS of these FIT-PNAs are shown in the Supporting Information (Figures S1−S8).…”

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

“…BisQ FIT-PNAs targeting the mutated KRAS oncogene DNA or mRNA had exceptional brightness (quantum yields Φ bound = 0.22-0.26) and showed selective fluorescence from Panc-1 cells (KRAS mutant) but not HT-29 or Bxpc-3 cells (KRAS wild type) [155]. The sequence context for BisQ fluorescence response has been examined thoroughly to help in the design of BisQ FIT-PNAs [259].…”