Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs

Abstract

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

Keywords: Ebola virus; Low-cost population screening; Oligonucleotide diagnostics; Point-of-care diagnostics; Rapid binding kinetics; Ratiometric fluorescence detection.