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

Accepted Manuscript Detection of unique ebola virus oligonucleotide sequence using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs Yijia Xiong, Tammie J. McQuistan, James W. Stanek, James E. Summerton, John E. Mata, Thomas C. Squier PII:

S0003-2697(18)30624-9

DOI:

10.1016/j.ab.2018.07.006

Reference:

YABIO 13074

To appear in:

Analytical Biochemistry

Received Date: 13 June 2018 Accepted Date: 12 July 2018

Please cite this article as: Y. Xiong, T.J. McQuistan, J.W. Stanek, J.E. Summerton, J.E. Mata, T.C. Squier, Detection of unique ebola virus oligonucleotide sequence using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs, Analytical Biochemistry (2018), doi: 10.1016/j.ab.2018.07.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Table of Contents Illustration

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Figure Legend: Increased binding selectivity and absence of charge density enables the selective detection of single or double stranded target oligonucleotides using phosphorodiamidate morpholino oligonucleotide (PMO) probes using fluorescence resonance energy transfer (FRET).

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Detection of Unique Ebola Virus Oligonucleotide Sequence using Fluorescently-Labeled Phosphorodiamidate Morpholino Oligonucleotide Probe Pairs

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Yijia Xiong,ǂ Tammie J. McQuistan,ǂ James W. Stanek,ǂ James E. Summerton,§ John E. Mata,ǂ,δ and Thomas C. Squier*,ǂ

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Department of Basic Medical Sciences, Western University of Health Sciences, 200 Mullins Drive, Lebanon, Oregon 97355, United States Gene Tools, LLC, One Summerton Way, Philomath, OR 97370, United States

Takena Technologies Inc., 405 West First Street, Albany, OR 97321, United States

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*Corresponding Author: Email: [email protected], Phone: (509) 531-4775

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ABSTRACT:

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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 ten minutes. 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.

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Key words: Ebola virus; oligonucleotide diagnostics; ratiometric fluorescence detection; lowcost population screening; point-of-care diagnostics; rapid binding kinetics.

ABBREVIATIONS:

EBOV, Ebola virus; EDTA, ethylenediaminetetraacetic acid; FRET, fluorescence resonance energy transfer; PCR, polymerase chain reaction; POC, point-of-care; PMO, phosphorodiamidate morpholino oligonucleotide; R0, Förster critical distance; TMR, tetrmethylrhodamine; and τ, time constant.

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INTRODUCTION:

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There is a strong need for the development of reagents that enable simple and fielddeployable surveillance of pathogens. The world health organization (WHO) and the non-profit Foundation for Innovative New Diagnostics (FIND) indicate a need for rapid pathogen screening that can be used by untrained staff, does not require electricity or can run on batteries or solar, and use reagents that can withstand temperatures of 40 °C [1]. In this respect, recent Ebola virus (EBOV) outbreaks highlight a need to develop new approaches suitable for population screening, with the goal of developing a general approach suitable for high-throughput array testing of a spectrum of different pathogens. Nonhuman primate models suggest that EBOV viral loads peak 5-7 days post infection, with viral loads approaching 1010 virus/mL (20 pM) [2]. The onset of symptoms commonly occurs 10 days following exposure [3], and is associated with a rapid decline of viral loads in both human populations and nonhuman primate (viral loads are typically about 108 /mL when symptoms develop) [3-5]. As IgG and IgM antibody titers are not detectable early in the infection, peaking at day 18 post-infection when viral titers fall below detectable levels [5], it is necessary to create assays that uniquely identify viral oligonucleotide targets. Current screening approaches, irrespective of available resources, emphasize nucleic acid amplification approaches or immunoassays that target the VP40 antigen in EBOV [6, 7]. An automated PCR instrument for EBOV diagnostics is the current state-of-the-science assay for point-of-care (POC) EBOV diagnostics [8]. However, the high cost of the instrumentation ($39,000) and indiviual assay ($189/sample), the long assay time (1 hour), and the inability to multiplex patient samples limit patient screening to cases following the appearance of symptoms (http://time.com/3544024/ebola-tests-fast-tracked-by-fda/). Critical to the disruption of disease progression is the development of low-cost population screening. Diagnostic assays that meet this requirment offer singular advantages, as peak viral loads occur in presymptomatic individuals when lower limits of nucleic acid detection are less stringent [2, 5].

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Current approaches that focus on EBOV RNA detection require nucleic acid amplification approaches (e.g., real-time PCR), which rely on the specificity and high-affinity binding characteristics of oligonucleotide primers (Kd < 1 × 10-11 M at 20 °C) [9-11]. Limitations are apparent even in clinical laboratories as PCR based approaches typically have poor sensitivity, with correct identification of viral infection less than 80% of the time [12-14]. The lack of sensitivity during POC implementation of diagnostics is commonly the result of inhibitors in complex biological samples (e.g., saliva) that interfere with target oligonucleotide isolation or result in a differential amplification efficiency of the target oligonucleotide relative to the standard curve target gene amplification efficiencies [15]. In comparison, immunoassays typically have an even lower sensitivity, with correct identification of viral infections about 50% of the time [1]. The lack of sensitivity using available antibodies is primarily the result of: i) cross-reactivity between different filovirus antigens and ii) sensitivities to antigen conformation that limit antibody recognition. In addition, current field-deployable antigen screening assays

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require cold-chains, with reagents kept between 2 °C and 8 °C [6], which represent significant barriers to implementation in low resource settings.

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Our focus is to develop low-cost and stable reagents that enable rapid detection of target oligonucleotides. To accomplish this goal, we exploit the ability of phosphorodiamidate morpholino oligonucleotide (PMO) probes to hybridize with target oligonucleotides under either low ionic strength conditions or in the presence of chaotropic agents (8 M urea). Low ionic strength conditions minimize nonspecific binding and disrupt target oligonucleotide secondary structures to enable predictable high-affinity binding [16, 17], circumventing common drawbacks of current diagnostics. Chaotropic agents act to lyse and inactivate pathogens to limit user exposure under conditions that stabilize RNA and other target oligonucleotides against nucleases. PMOs are synthesized with an available amine at the 5’ or 3’ positions, allowing simple conjugation with amine-reactive fluorophores (Figure S1). Following derivatization of PMO probe pairs with fluorescence donor or acceptor fluorophores capable of undergoing fluorescence resonance energy transfer (FRET) (Figure 1), PMO probe pairs provide a pathforward to create the high-affinity capture and ratiometric detection capabilities needed to identify pathogens in complex biological fluids. PMO probes overcome problems associated with the use of other nucleotide analogs, such as phosphorothioates (S-DNA), while retaining nuclease resistance and binding specificity [16]. As a result, PMOs remain intact for many hours in extracellular fluids (blood and saliva) and cellular lysates, and unlike other oligonucleotides the hybridization of PMOs with RNA does not activate RNase H-cleavage mechanisms that can be problematic for common RT-PCR methods that only extrapolate initial target oligonucleotide concentrations. Using PMO probe pairs in combination with ratiometric fluorescence detection, we are able to directly detect unique 70 base-pair oligonucleotide targets derived from the EBOV genome with a lower detection limit of 25 pM with no affinity isolation or nucleic acid concentration steps. Using simple affinity columns to enrich target oligonucleotides, lower detection limits can be reduced by several orders of magnitude to enable the routine detection of EBOV viral loads in pre-symptomatic individuals [5].

Figure 1: PMO Probe Pair Design for Diagnostic Detection of Target Oligonucleotides. Schematic (Panel A) and nucleotide sequences (Panel B) of Alexa488-labeled and tetramethylrhodamine (TMR) labeled PMO probe pairs showing complementarity with target oligonucleotide and proximity between donor (Alexa Fluor 488) and acceptor (TMR) chromophores for PMO probe pairs bound to target oligonucleotide to enable fluorescence resonance energy transfer (FRET).

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MATERIALS AND METHODS:

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Alexa Fluor 488 5-TFP (A30005), Alexa Fluor 546 succinimidyl ester (A20002), and tetramethylrhodamine isothocyanate (TMR) (T490) were purchased from Molecular Probes ThermoFisher Scientific (Eugene, OR). Fluorescein isothiocyanate was obtained from SigmaAldrich (St. Louis, MO). Target DNA (200 nmol aliquots) and the complementary sequence (used as a control) were purchased from ThermoFisher Scientific (Waltham, MA). Target DNA sequences correspond to a 70 base-length highly conserved and unique region in the EBOV RNA sequence (5’-CTG GGACCGGACTGCTGTATGGAACCACATGATTGGACCAAGAACAT AACAGAC-3’; mass = 16,692 Da), which was chosen based on a Blast search against all EBOV variants published in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), which contain approximately 19,000 nucleotides [18, 19]. DNA or PMO probes, corresponding to unique 25 nucleotide sequences complementary to the target oligonucleotide, were obtained from either Invitrogen (DNA probes) or Gene Tools, LLC (PMO probes), with amines incorporated at either the 5’ position (upstream probes) or 3’ position (down-stream probes) (Figure 1; Figure S1). Pooled normal human saliva was from Innovative Research (IR100044P) (Novi, MI). Following removal of mucus (Zebra Spin Desalting Column, 7 kDa MWCO; ThermoFisher Scientific) and cellular disruption (8.0 M urea), the nucleotide concentrations in the isolated saliva was 0.3 mM (http://www.sigmaaldrich.com/technical-documents/articles/biology/quantitation-of-oligos.html).

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Functionalization and Chromatographic Separation of PMO and DNA Probes. Fluorescent dyes (2 mM) were conjugated with either DNA or PMO probes (11 µM) in 0.1 M Na2HCO3 (pH 8.3) and 10% (v/v) DMSO for 16 hours at 22 °C (10 rpm) in the dark. In the case of PMO oligonucleotides, the separation of fluorescently-labeled probes from excess dye required a combination of size exclusion and anion exchange chromatography. Labeled oligonucleotides were first applied to a Superdex 75 10/300 GL size exclusion column (GE Heathcare Life Sciences, Pittsburgh, PA), in which sample was eluted in 10 mM Tris (pH 8.0) and 1 mM EDTA (TE buffer). Fractions containing the labeled PMO probe were then pooled and subsequently purified using a HiTrap Q HP anion exchange column (5 mL) (GE Healthcare Life Sciences, Pittsburgh, PA) in 10 mM NaOH (pH 12.0) using a salt gradient from 0 M to 1 M NaCl. In the case of DNA oligonucleotides, size exclusion chromatography was sufficient to separate fluorescently labeled DNA from free dye. Determination of Oligonucleotide Labeling Stoichiometries. Oligonucleotide probe concentrations were determined using calculated extinction coefficients (ε260) provided by the manufacturer, which for CCCTGGCCTGACGACATAGCTTGGT were 251,220 M-1 cm-1 (PMO) and 258,800 (DNA) and for CTAACCTGGTTCTTGTATTGTCTGT were 250,740 M-1 cm-1 (PMO) and 259,500 M-1 cm-1 (DNA). Following oligonucleotide probe derivatization with either tetramethylrhodamine (TMR) or Alexa Fluor 488, concentrations of oligonucleotides were determined using their molar extinction coefficients following correction for dye absorbance at 260 nm, which for TMR was 0.1 x Abs555nm and for Alexa Fluor 488 was 0.3 x Abs495nm. Concentrations of tetramethyl-rhodamine (TMR) or Alexa FluorTM 488 were determined using

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ε555 = 80,000 M-1 cm-1 and ε495 = 73,000 M-1 cm-1 (https://www.thermofisher.com). Stoichiometries of labeling were determined to be 0.9 ± 0.1 dyes bound per DNA or PMO probe for either the Alexa Fluor 488 or TMR dyes.

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Fluorescence Spectroscopy. Steady-state fluorescence measurements were acquired using a FluoroMax-4 spectrofluorometer (Horiba Scientific, Newton, NJ) equipped with a Xenon lamp. Excitation was at 470 nm and fluorescence emission was collected using 5 nm slit widths for both excitation and emission.

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Determination of Binding Affinities. Unless otherwise specified, binding affinities between oligonucleotide probes and target DNA were measured in 10 mM Tris (pH 7.5) and 1.0 mM EDTA using changes in fluorescence intensity associated with either the Alexa Fluor 488 or TMR chromophores that were respectively bound to the upstream or downstream oligonucleotide probe. Typically, this involves the titration with either the target oligonucleotide or an acceptor DNA or PMO probe (ligand), enabling the concentration of the reporter chromophore on either the donor or acceptor oligonucleotide probe to be kept at a fixed concentration (Figure 3). The binding affinity (Kd) was determined assuming a Langmuir binding isotherm, where changes in fluorescence intensity (∆F / ∆Fmax) of the reporter chromophore (typically Alexa Fluor 488; λem = 517 nm) on the probe is used to monitor binding to the target oligonucleotide, where:

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Virtually identical binding affinities (Kd) were obtained using the equivalent model expressed as a quadratic using total amounts of added ligand and reporter (Figure S3), where:

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&'()*+,- .,/-0 1[2+3(-0]4 + [567,'*6']4 + 89 : − ; 1[2+3(-0]4 + [567,'*6']4 + 89 :< − 4 × [2+3(-0]4 × [567,'*6']4 = 2 × [567,'*6']4

Determination of Bimolecular Rate Constants. Alexa Fluor488-labeled probes were hybridized with an equimolar concentration of oligonucleotide target (0.3 µM) in 10 mM TRIS (pH 7.5), 1.0 mM EDTA, and 150 mM NaCl (TES buffer) for 16 hours at 22 oC. Samples were then diluted and equimolar amounts of the TMR-labeled oligonucleotide probe was added to the preformed complex between the Alexa Fluor488-labeled probe and oligonucleotide target in the presence of either 5 mM (low salt) or 150 mM (high salt) NaCl. Bimolecular binding constants between oligonucleotide probes and target DNA were determined by fitting time-dependent changes in the fluorescence intensity of Alexa Fluor 488 to a second-order rate equation, where:

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∆& 1 = * , ∆&?@A 1 + C 1 = D [E('36*]. C

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The LOD was determined to be 25 pM.

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Determination of Limit of Detection. The limit of detection (LOD) for a linear calibration curve is determined with 99% confidence by three times the standard deviation of the y-intercept (σ) over the slope of the calibration curve [20], where:

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RESULTS AND DISCUSSION:

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Design of PMO Probe Pairs. Phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs (25-bases in length) were designed to bind to unique target sequences within the EBOV genome, such that covalently-bound fluorescent dyes bound to each of the probe pairs are brought into close proximity upon binding target oligonucleotides, enabling detection through fluorescence-resonance energy transfer (FRET). To accomplish this, the 5’ end of the upstream PMO probe was labeled with Alexa Fluor 488, which serves as a FRET donor (D), while the 3’ end of the downstream PMO probe was labeled with tetramethylrhodamine (TRM), which functions as a FRET acceptor (A)(Figure 1). A three-nucleotide gap between PMO probe pairs minimizes possible steric interference associated with the dyes. In designing this gap, we note that prior measurements have established that gaps of up to four nucleotides in length have a minimal effect on the conformation and dynamics of double-stranded DNA [21]. Similar constructs were made using DNA probe pairs, enabling a comparison of binding and kinetics of conventional and PMO probe pairs.

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Fluorescence Labeling and Purification of PMO Probe Pairs. Near equimolar stoichiometric labeling of PMO oligonulceotide probe pairs (11 µM) by either tetramethylrhodamine isothiocyanate (TMR) or Alexa Fluor 488 5-TFP ester required addition of a 180-fold molar excess of dye. Separation of free (unreacted dye) from the dye-labeled oligonucleotide conjugate was accomplished using orthogonal chromatographic separations. First, size-exclusion chromatography using a Superdex 75 10/300 GL resin was used to enrich the fluorescentlylabeled 25 base-pair PMO probes from both free dyes and unmodified oligonucleotides, where the latter come off the column at larger elution volumes due to their lower masses. Irrespective of whether TMR or Alexa Fluor 488 chromophores were used, in all cases we observe that the unbound dye eluted over a large range of elution volumes under conditions involving either oligonucleotide probe labeling or under control experiments with no added oligonucleotide (dye only). In the case of the TMR-labeled PMO probe, fractions were collected that eluted between 13.0 and 14.5 mL (Figure 2A). These fractions were pooled and further purified from unbound TMR using a HiTrap Q HP anion exchange column under alkaline conditions (pH 12) that imparts a negative charge on the morpholino backbone (Figure 2B). We collected fractions that elute between 44 mL and 53 mL (Figure 2B); the stoichiometry of TMR probe bound to the PMO probe was 0.9 ± 0.1 dye bound per PMO. Similar chromatographic separations and stoichiometries of labeling are observed for the Alexa Fluor 488-bound PMO probe following the same orthogonal separations (Figure S2). High-Affinity Binding Between PMO Probe Pair and Oligonucleotide Target. Despite the widespread use of PMOs for a range of gene silencing applications [22-27], there have been no prior reports of the binding affinities between PMO probes and target oligonucleotides. We have, therefore, measured the binding affinities between Alexa488- and TMR-labeled PMO probes and an oligonucleotide identified using a BLAST search that is homologous to a unique sequence within the Ebola virus [18]. We focus on using a DNA oligonucleotide target for two reasons.

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First, the use of a DNA target for early stage design of a diagnostic assay limits possible artifacts associated with the lower stability of RNA. Second, and more importantly, there are no current assays that enable screening of host genomes for integrated copies of EBOV, which have been suggested to serve as pathogen reservoirs [28, 29].

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Figure 2: Purification of Fluorescently Labeled PMO Probes. Sequential separation of PMO probe (CTAACCTGGTTCTTGTATTGTCTGT) labeled with tetramethylrhodamine (TMR) from unbound dye through size exclusion (panel A) and anion exchange (panel B) chromatography. TMR-PMO was separated from the majority of unbound dye using a Superdex 75 10/300 GL size exclusion column (void volume < 9 mL) in 10 mM Tris (pH 8.0) and 1 mM EDTA; absorbance was monitored at 280 nm (solid black line) and 557 nm (blue dashed line) (Panel A). Fractions collected between 13.0 and 14.5 mL (blue bar) were further separated using a HiTrap Q HP anion exchange column (5 mL) (GE Healthcare Life Sciences, Pittsburgh, PA) in 10 mM NaOH (pH 12.0) using a salt gradient from 0 M to 1 M NaCl; absorbance at 280 nm (solid black line) and conductivity (red dashed line) were monitored (panel B). Purified TMR-PMO was collected in fractions between 44 mL and 53 mL (red bar). Stoichiometry of TMR bound to PMO probe was calculated to be 0.9 ± 0.1 dyes bound per PMO probe (n = 2), as described in Experimental Procedures.

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Binding between the PMO probe and the target oligonucleotide was monitored using the known environmental sensitivity of Alexa Fluor 488 [30]. which results in a two-fold increase in the fluorescence quantum yield upon binding target oligonucleotide (Figure 3A). There is a progressive increase in the fluorescence of the Alexa488-PMO probe upon addition of target oligonucleotide, which can be fit assuming a Langmuir binding isotherm (Kd = 0.31 ± 0.04 nM)(Figure 3C). A very similar high-affinity binding was observed using fluorescence changes of the TMR-PMO probe upon binding target oligonucleotides (Kd = 0.18 ± 0.02 nM; see Figure S3). These results indicate that no significant differences arise in binding affinities as a result of sequence differences, including differences in G:C base pairing (i.e., 15 G:C base-pairs using Alexa488-PMO probe versus 10 G:C base-pairs using TMR-PMO probe). These results are consistent with suggestions that enhanced stacking between adjacent bases following probe binding to complementary DNA targets are largely responsible for the stability of the double helix [21]. However, the measured binding affinity between the 25-base PMO probe and the target DNA is an order of magnitude lower than that observed using similar length DNA probes, where Kd < 10 pM [11, 31, 32]. Reductions in binding affinities may be related to the 9

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conformational dynamics of the PMO backbone, as prior results have demonstrated that reductions in the charge on the phosphate backbone reduce the conformational flexibility of the DNA duplex [33], which has the potential to modify intramolecular torsional parameters that may restrict optimal base stacking [34]. From a practical point of view, the lower binding affinity of the PMO probes enhances selectivity, as longer regions of complementarity are needed for high-affinity binding between PMO probes and complementary oligonucleotides [27, 35].

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Figure 3: High-affinity Binding of PMO Probes to Target Oligonucleotides. Fluorescence emission spectra (panels A and B) and binding curves (panels C and D) for Alexa488-PMO (1.0 nM) alone or in the presence of variable amounts of target oligonucleotide (panels A and C); alternatively, spectra and associated binding curve are shown for Alexa488-PMO in complex with target oligonucleotide (0.1 nM) in the presence of variable amounts of TMRPMO (panels B and D). Buffer conditions include 10 mM Tris (pH 7.5) and 1 mM EDTA. [Target] was 0 nM (red), 0.125 nM (blue), 0.5 nM (magenta), and 4 nM (black) (Panel A). [TMR-PMO] was 0 nM (black), 1.0 nM (red), 2.0 nM (magenta), 2.8 nM (blue), and 16 nM (navy) (Panel B). Binding curves (Panels C and D) represent nonlinear least squares fits to the data, as described in experimental procedures, where Kd (Alexa488-PMO) = 0.31 ± 0.04 nM (Panel C) and Kd (TMR-PMO) = 1.2 ± 0.2 nM (Panel D). λex = 470 nm; fluorescence signals were corrected for Raman scattering.

Fluorescence Resonance Energy Transfer Between Alexa488-PMO and TMR-PMO Probes. Increased assay selectivity is proposed to result from the assay design, as detection requires association of both Alexa488-PMO and TMR-PMO probes to target oligonucleotides to bring donor and acceptor chromophores into close proximity to enable FRET (Figure S4) [36]. Using changes in the FRET efficiency, we have measured the binding affinity of the TMR-PMO probe to a stoiochiometric complex between Alexa488-PMO and target oligonucleotide (Figure 3B). Upon increasing the concentration of the TMR-PMO probe there is a progressive decrease in the fluorescence intensity of the donor (i.e., Alexa Fluor 488) peak at 517 nm, with corresponding 10

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increases in the fluorescence intensity of the acceptor (i.e., TMR) peak at 577 nm due to FRET. Decreases in the fluorescence intensity (∆F) of the Alexa Fluor 488 chromophore were used to monitor binding of the TMR-PMO probe to the complex with Alexa488-PMO and target oligonucleotide, where the fraction bound equals ∆F/∆Fmax (Figure 3D). Fitting the binding curve to a Langmuir isotherm yields a binding affinity (Kd = 1.2 ± 0.2 nM), which is weaker than that associated with the binding of either the Alexa488-PMO (Kd = 0.31 ± 0.04 nM) or TMRPMO (Kd = 0.18 ± 0.02 nM) probe alone (Figure 3C; Figure S2). These results suggest an anticooperative binding mechanism, whereby binding of the first probe (i.e., Alexa488-PMO) modifies the target oligonucleotide structural dynamics in such a way that association of the second probe (i.e., TMR-PMO) is reduced. These results support a model in which the target oligonucleotide adopts a highly structured tertiary structure upon binding one probe, acting to restrict the dynamics of the oligonucleotide target to reduce the subsequent binding affinity of the second probe pair.

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Binding Kinetics Between PMO Probe and Target Oligonucleotide. To identify optimal conditions for rapid target hybridization, we have assessed the effect of ionic strength on the binding kinetics between DNA or PMO probes and target oligonucleotides. Under high-salt conditions (i.e., 0.15 M NaCl), we measured rates of hybridization upon addition of the FRET acceptor probe (i.e., TMR-PMO or TMR-DNA) to an equimolar concentration of a preformed complex between the FRET donor probe (i.e., Alexa488-PMO or Alexa488-DNA) and the target oligonucleotide. Hybridization was measured as the time-dependent decrease in the fluorescence intensity of the donor probe at 517 nm (Figure 4).

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Figure 4: Increased Rates of Target Oligonucleotide Binding using PMO Probes. Hybridization kinetics upon addition of TMR-labeled DNA (Panel A) or PMO probes (Panels B and C) to a preformed complex between an Alexa488-labeled probe and target oligonucleotides. Experiments used equimolar concentrations of each probe pair and target oligonucleotide, which were varied between 0.5 nM (black), 1.0 nM (red), 2.0 nM (green), 3.5 nM (cyan), and 5.0 nM (blue) in 10 mM Tris (pH 7.5) and 1 mM EDTA in the presence of either high salt (150 mM NaCl; Panels A and B) or low salt (5 mM NaCl; Panel C). Second order rate constants were obtained from the concentration-dependence of the hybridization rates (1/τ) for TMR-labeled DNA (black squares) or PMO (red or blue circles) probe binding to target oligonucleotides in complex with Alexa488-labeled probes, where ∆F = 1/(1 + t/τ), which were 4.4 ± 0.4 × 105 M-1 s-1 (DNA probe, high salt), 4.8 ± 0.5 × 105 M-1 s-1 (PMO probe, high salt), and 15 ± 2 × 105 M-1 s-1 (PMO probe, low salt). Hybridization kinetics followed decreases in the fluorescence emission of the FRET donor Alexa488-labeled donor probes upon binding of the FRET acceptor TMR-labeled probe to the oligonucleotide target. λex = 470 nm; λem = 515 nm.

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Similar hybridization rates (1/τ) are observed using either PMO or DNA probes, which increase from about 0.03 min-1 to about 0.16 min-1 upon increasing the oligonucleotide concentration from 0.5 nM to 5.0 nM. Second-order bimolecular rate constants were obtained from the concentration-dependence of the hybridization rates (Figure 4D), which were 4.4 ± 0.4 × 105 M-1 s-1 (DNA probe) and 4.8 ± 0.5 × 105 M-1 s-1 (PMO probe). These second-order rate constants are consistent with prior association rate constants between complementary oligonucleotides of DNA that, depending on the length of the oligonucleotide, range from 4 x 105 to 2 x 107 M-1 sec-1 [9, 37]. PMO probes have no net charge, as the nucleic acid bases in PMO probes are connected to the backbone through morpholine rings, which are linked together through bulky phosphorodiamidate groups instead of phosphates (Figure S1). In comparison, DNA probes are highly charged, and the nucleic acid bases are connected to the backbone through deoxyribose sugars linked together through phosphodiester bonds. The observation that DNA and PMO probes have very similar hybridization rates, using the same complementary DNA strand, suggests that differences in the structure and charge of the PMO and DNA probe backbones have minimal effects on rates of binding under these conditions of high ionic strength (i.e., 0.15 M NaCl). We note that under these high-salt conditions, the total charge density on the ssDNA is screened, acting to increase the flexibility of the backbone to enhance the formation of more compact and folded structures (e.g., stem-loops) [38, 39], which can interfere with the hybridization between two complementary DNA strands [40].

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Complementary DNA oligonucleotides do not hybridize under conditions of low ionic strength due to electrostatic repulsion (Figure S5). In contrast, PMO probes have no net charge and bind to ssDNA target oligonucleotide with high-affinity even under conditions of low ionic strength (Figure 3; Figure S6). We have measured rates of TMR-PMO binding to a preformed complex between Alexa488-PMO and target oligonucleotide (Figure 4C). In comparison to the same experiment done under conditions of high ionic strength there is a three-fold increase in the binding kinetics; the second-order bimolecular rate constant obtained from the concentrationdependence of the hybridization rates is 15 ± 2 × 105 M-1 s-1 (Figure 4D). The three-fold increase in the rate of binding is consistent with the disruption of stem-loop structures in the ssDNA target under conditions of low ionic strength (5 mM NaCl) due to electrostatic repulsion. Indeed, calculations suggest that about 20% of the EBOV target oligonucleotide undergoes base pairing under conditions of high ionic strength [41]. Target Oligonucleotide Detection. Upon binding target oligonucleotides there is FRET between donor Alexa488-PMO probes (λem = 517 nm) and acceptor TMR-PMO probes (λem = 577 nm) that results in a decrease in donor fluorescence and an increase in acceptor fluorescence (Figure 3B). To enable the development of a sensitive and reliable detection platform, we have measured the ratiometric change (∆R) of the fluorescence signal of the acceptor TMR-PMO probe over that of the donor Alexa488-PMO probe in the presence of variable amounts of the target oligonucleotide (Figure 5) [42]. There is a linear relationship in this FRET signature, with a lower detection limit (LDL) of 25 pM [20]. This is significant, as peak viral loads of EBOV 12

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Figure 5: Lower-Limit of Target Oligonucleotides Detection. Observed linear dependence between target oligonucleotide concentration and ratiometric change (∆R) in TMR acceptor fluorescence (Fl) [λem(max) = 577 nm ] relative to Alexa Fluor 488 donor Fl [λem(max) = 517 nm], where ∆R = ∆ [Fl577(TMR)/Fl517(Alexa Fluor 488)]. Experimental conditions include 1 nM Alexa488-PMO, 1 nM TMR-PMO, and variable amounts of target oligonucleotide in 10 mM Tris (pH 7.5) and 1 mM EDTA. λex = 470 nm. Lower detection limit of target oligonucleotide is 25 pM, where ∆R = 0.149 ± 0.001 × [Target] in nM.

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Lower detection limits are possible upon the inclusion of routine oligonulceotide enrichment and clean-up procedures (which obtain 10-20 fold enrichments of viral RNA over host background) [43], which can extend the capability of the assay to enable low-cost and highthroughput population screening when viral loads are around 0.2 pM (which commonly occurs at the onset of symptoms) [5, 44-46]. A simple and direct assay is important, as reliable RNA isolation of virus from tissues remains problematic, making it challenging to obtain accurate viral loads using nucleic acid amplification approaches [44]. Furthermore, as EBOV loads are highly correlated with patient outcomes and the progression of epidemics [46], it is important to have high-throughput and field deployable approaches that overcome current limitations involving nucleic acid amplification approaches, where individual assays typically require 60 minutes using expensive intrumentation [47].

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Detection of Single and Double Stranded Target DNA. DNA and PMO probe pairs are able to detect single stranded oligonucleotide targets under high ionic strength conditions (150 mM NaCl) with similar sensitivities, which is apparent from the similar reductions in the fluorescence emission of the Alexa Fluor 488 donor probes (λem = 517 nm) and similar increases in TMR acceptor probes (λem = 577 nm) (Figure 6A, B). Under these high-salt conditions double stranded DNA target cannot be detected, as the complementary strands quickly rehybidize following transient heating for two minutes at 95 °C due to their higher affinity constants relative to the shorter DNA or PMO probe pairs. However, while electrostatic repulsion prevents the DNA probe pairs from binding at low ionic strength (5 mM NaCl), the PMO probe pairs retain their high-affinity binding irrespective of the ionic strength.

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Figure 6: Sensitive Detection of Both Single-Stranded and Double-Stranded Oligonulcleotide Target Sequences Using PMO Probe Pairs. Fluorescence emission spectra for DNA (Panel A; 10 nM) or PMO (Panels B and C; 10 nM) probe pairs upon incubation with single-stranded (blue lines) or double-stranded (black lines) target oligonucleotides (10 nM) in 10 mM Tris (pH 7.5) and 1.0 mM EDTA in the presence of high (150 mM; panels A and B) or low (5 mM; panel C) amounts of NaCl. λex = 470 nm. Ratiometric changes (∆R) in the fluorescence emission spectra resulting from probe-pair binding to target oligonucleotides are depicted for both single-stranded (Panel D) and double-stranded (Panel E) oligonucleotide target. Experimentally, double-stranded oligonucleotide target was hybridized upon incubation of target strand (300 nM) with complementary strand (300 nM) overnight in 10 mM Tris (pH 7.5), 1 mM EDTA, and 150 mM NaCl at 25 ºC. Spectra were taken following transient heating for two minutes at 95 °C.

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Following transient heating for two minutes at 95 °C it is apparent that the fluorescence emission spectra demonstrates a reduction in the fluorescence emission of the Alexa488 donor probes (λem = 517 nm) and increase in TMR acceptor probes (λem = 577 nm) that is virtually identical to that observed using single-stranded oligonucleotide targets (Figure 6C). These results demonstrate a utility of the PMO probes to assess for the presence of double stranded DNA targets [48]. Such approaches will enable screening for the possible integration of EBOV into host genomes, which is important given suggestions that the integration of EBOV into host genomes represents an important viral reservoir [28, 29].

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Selective Detection of Target Oligonucleotides in Human Saliva. To assess the possible offtarget binding of PMO probe pairs to oligonucleotides present in biological samples, we have assessed the binding of the PMO probe pairs to target oligonucleotides in the absence and presence of human saliva (Figure 7). In these experiments, the probe pair and oligonucleotide target are present at a low concentration (1.0 nM), such that any nonspecific PMO probe binding will reduce FRET between the Alexa488-PMO donor and the TMR-PMO acceptor probes. Changes in the fluorescence emission spectra are assessed upon addition of the target oligonucleotide (1.0 nM) in buffer (Figure 7A) or in the presence of human saliva (Figure 7B). Chaotropic agents (i.e., 8 M urea) were added to disrupt cells in the saliva.

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In the absence of oligonucleotide target the fluorescence emission spectra are representative of the Alexa488-PMO donor; there is no FRET to the TMR-PMO acceptor. Virtually identical fluorescence emission spectra are observed in buffer or in the presence of human saliva upon addition of the target oligonucleotide, with significant reductions in the fluorescence emission of the Alexa488-PMO donor (λem = 517 nm) and increases in the TMR-PMO acceptor (λem = 577 nm), indicating that very similar amounts of FRET occur. These results demonstrate that there is no off-target binding of the PMO probes, as this would act to reduce the observed FRET. This is significant given the large excess of oligonucleotides in the saliva (0.3 mM nucleotide bases), which represents a greater than 4,000-fold excess of nucleotide bases in the saliva relative to the target oligonucleotide (70 nM nucleotide bases).

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Figure 7: Minimal Off-Target Binding for PMO Probes. Fluorescence emission spectra for PMO probe pairs (1 nM) in buffer (Panel A) or human saliva (Panel B) in the absence (black dashed lines) or presence (solid colored lines) of target oligonucleotide (1 nM) in 10 mM Tris (pH 7.5), 1 mM EDTA, and 8 M urea. λex = 470 nm.

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In summary, we have demonstrated that PMO probe pairs rapidly hybridize with target oligonucleotides under low ionic strength conditions or in the presence of chaotropic agents (Figures 4 and 7). These experimental conditions minimize nonspecific binding and disrupt the formation of secondary structures within target oligonucleotide (Figure 7) [16, 17], which enable the confident design of high-affinity probes to detect either single or double stranded oligonucleotides within complex biological samples (e.g., saliva). Using a ratiometric fluorescence signal to detect the presence of EBOV (Figure 5), our approach overcomes prior limitations involving the use of PMO probes in the development of diagnostic assays (http://www.gene-tools.com/diagnostics), which typically require calibration curves of known standards that limit field utility or lack the needed sensitivity for clinical pathogen detection [17, 49, 50]. Upon target binding, observed changes in FRET provide a needed sensitivity for pathogen detection with a 25 pM lower limit of detection (Figure 5); no off-target binding is apparent within a complex mixture of nucleic acids and other biomolecules present in human saliva samples (Figure 7). Equivalent levels of FRET are achieved using PMO probes for single or double stranded DNA targets (Figure 6); in comparison FRET signals using DNA probes with the same donor and acceptor probes are insensitive to the presence of double stranded target nucleic acids. In conclusion, our results indicate an ability to use PMO probes to directly detect clinically relevant levels of oligonucleotide targets in biological samples without the need for 15

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nucleic acid amplification, and open the possibility of screening for genome integration of DNA based copies of viral RNA.

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APPENDIX. SUPPLEMENTARY DATA:

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Additional data are available regarding the structures of the PMO probe pairs (Figure S1), purification of Alexa488-labeled PMO probe (Figure S2), binding affinities of Alexa488-PMO and TMR-PMO probes for target oligonucleotides (Figure S3), calculated energy transfer efficiences between Alexa488-PMO and TMR-PMO bound to target oligonucleotide (Figure S4), fluorescence emission spectra for DNA probe pairs in the presence of target oligonuleotides (Figure S5), and fluorescence emission spectra for PMO probe pairs in the presence of target oligonucleotides (Figure S6). This material is available free of charge.

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AUTHOR INFORMATION: Corresponding Author

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*(541) 259-0230. E-mail: [email protected]. FAX: (541) 259-0201.

The authors declare no competing financial interests.

ACKNOWLEDGMENTS:

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This work was supported by the John C. Erkkila M.D. Endowment for Heath and Human Performance. We thank Dr. Hong M. Moulton for insightful discussions. We thank Ceili Smythe and Jocelyn Powell for technical assistance.

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Figure Legend: Increased binding selectivity and absence of charge density enables the selective detection of single or double stranded target oligonucleotides using phosphorodiamidate morpholino oligonucleotide (PMO) probes using fluorescence resonance energy transfer (FRET).

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Supplementary Data for

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Detection of Unique Oligonucleotide Sequences Present in the Ebola Virus using Fluorescently-Labeled Phosphorodiamidate Morpholino Oligonucleotide Probe Pairs

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Yijia Xiongǂ, Tammie McQuistanǂ, James W. Stanekǂ, James E. Summerton§, John E. Mataǂ,δ, and Thomas C. Squier*,ǂ

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Department of Basic Medical Sciences, Western University of Health Sciences, 200 Mullins Drive, Lebanon, Oregon 97355, United States Gene Tools, LLC, One Summerton Way, Philomath, OR 97370, United States

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Takena Technologies Inc., 405 West First Street, Albany, OR 97321, United States

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Figure S6: Fluorescence Emission Spectra for PMO Probe Pairs in the Presence of Target Oligonucleotides …………….…………..

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Figure S1: Structures of PMO Probe Pairs. Schematic depiction (Panel A), structures (Panel B), and nucleotide sequence (Panel C) of Alexa488- and TMR-labeled PMO probe pairs. PMO probes were designed with a 5’ amine to enable conjugation with the dye Alexa Fluor 488 or a 3’ amine to enable conjugation with dye tetramethylrhodamine (TMR). Alexa Fluor 488 and TMR respectively function as donor or acceptor chromophores for fluorescence resonance energy transfer (FRET) measurements. FRET occurs upon PMO probe pair binding to target oligonucleotides, which acts to bring these dyes into close proximity (i.e., distance < 0.5 × R0, where R0 = 6.2 nm [51].

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Figure S2: Purification of Alexa488-labeled PMO Probe. Chromatograms showing the sequential separation of PMO probe (CCCTGGCCTGACGACATAGCTTGGT) labeled with Alexa Fluor 488 from unbound dye. Alexa488-PMO was separated from the majority of unbound dye using a Superdex 75 10/300 GL size exclusion column (void volume < 9 mL) in 10 mM Tris (pH 8.) and 1 mM EDTA; absorbance was monitored at 280 nm (solid black line) and 557 nm (dashed blue line) (Panel A). Fractions collected between 11.4 mL and 13.2 mL (blue bar) were further separated using a HiTrap Q HP anion exchange column (5 mL) (GE Healthcare Life Sciences, Pittsburgh, PA) in 10 mM NaOH (pH 12) using a salt gradient from 0 M to 1 M NaCl; absorbance at 280 nm (solid black line) and conductivity (dashed red line) were monitored (panel B). Fractions between 44.9 mL and 51.9 mL (red bar) were collected and used.

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Figure S3: Binding Affinities of Alexa488-PMO and TMR-PMO Probes for Target Oligonucleotides. Binding curves for Alexa488-PMO (Panel A) or TMR-PMO (Panel B) using 0.1 nM of either probe in the presence of variable amounts of target oligonucleotide in 10 mM Tris (pH 7.5) and 1 mM EDTA. Symbols represent averages and standard deviations (n = 3). Lines represent least squares fits to the data using a one-site binding model, where Kd (Alexa488-PMO) = 0.24 ± 0.01 nM and Kd (TMR-PMO) = 0.18 ± 0.02 nM. Fluorescence emission spectra were corrected for Raman scattering prior to analysis. Upon target oligonucleotide binding, there was a 1.6-fold and 5.6-fold increase in the respective fluorescence of Alexa488-PMO or TMR-PMO. λex = 470 nm for Alexa Fluor 488 and 532 nm for TMR.

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Figure S4: Calculated Energy Transfer Efficiency Between Alexa488-PMO and TMR-PMO Bound to Target Oligonucleotide. Fluorescence emission spectra in the presence of target oligonucleotide (10 nM) for the donor Alexa Fluor 488-PMO (10 nM; green), the acceptor TMRPMO (10 nM; orange), and in the presence of both Alexa488-PMO and TMR-PMO probes (navy blue) in 10 mM Tris (pH 7.5), 1.0 mM EDTA and 150 mM NaCl. The fluorescence resonance energy transfer (FRET) efficiency was calculated to be 55%, suggesting that the labeling of each PMO probe was incomplete. FRET efficiences were determined by assuming a linear combination of spectra for the donor or acceptor probe bound to target oligonucleotide, where the observed spectrum equals 0.45 × donor spectrum + 3.55 × acceptor spectrum (calculated spectrum is pink dashed line).

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Figure S5: Fluorescence Emission Spectra for DNA Probe Pairs in the Presence of Target Oligonucleotides. Experimental spectra for sample containing Alexa488-labeled DNA (10 nM), Alexa546-labeled DNA (10 nM), and target oligonucleotide (10 nM) (red lines) in comparison to expected spectrum in the absence of FRET (blue lines), which represents a digital summation of the fluorescence emission spectra for Alexa488-labeled DNA (10 nM) or Alexa546-labeled DNA (10 nM) that were obtained individually in the presence of target oligonucleotide (10 nM). Spectra were obtained in 10 mM Tris (pH 7.5) and 1 mM EDTA under low salt conditions (no added NaCl) (Panels A and B), high salt conditions (150 mM NaCl) (Panels C and D), or in the presence of 8 M urea (Panels B and D). The sequence of the Alexa546-labeled DNA probe was modified to reduce the nucleotide spacing between donor and acceptor probes by one nucleotide (i.e., 5’ GTCTGTTATGTTCTTGGTCCCAATCA 3’). Fluorescently-labeled DNA probe pairs were purified from unbound dye following size-exclusion chromatography.

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Figure S6: Fluorescence Emission Spectra for PMO Probe Pairs in the Presence of Target Oligonucleotides. Fluorescence emission spectra for sample containing fluorescein-PMO (10 nM), Alexa546-PMO (10 nM), and target oligonucleotide (10 nM) (red lines) in comparison to expected spectrum in the absence of FRET (blue lines), which represents a digital summation of the fluorescence emission spectra for fluorescein-PMO (10 nM) or Alexa546-PMO (10 nM) that were obtained individually in the presence of target oligonucleotide (10 nM). Spectra were obtained in 10 mM Tris (pH 7.5) and 1 mM EDTA under low salt conditions (no added NaCl) (Panels A and B), high salt conditions (150 mM NaCl) (Panels C and D), or in the presence of 8 M urea (Panels B and D). Fluorescein-labeled PMO probe sequence was identical to that used for Alexa488-PMO (i.e., 5’ TGGTTCGATACAGCAGTCCGGTCCC 3’). The sequence of the Alexa546 PMO probe was modified to reduce the nucleotide spacing between donor and acceptor probes by one nucleotide (i.e., 5’ GTCTGTTATGTTCTTGGTCCCAATCA 3’). PMO probe pairs were isolated following size-exclusion chromatography without a second step involving an anion exchan.ge column to remove unbound dye, resulting in a significant background signal.

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Fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) can be purified at pH 12 Hybridization with target oligonucleotides occurs within ten minutes Picomolar lower-limit-of-detection enables point-of-care screening for presence of Ebola virus at levels seen in presymptomatic individuals Binding under low-ionic strength conditions enables routine detection of single-stranded or double-stranded oligonucleotide targets PMO probe pairs enable sensitive detection with no off-target binding

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