Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) withPeptide Nucleic Acid Hybridization ProbesLiang Hu,† Mansi Anand,† Svetlana M. Krylova,† Burton B. Yang,‡ Stanley K. Liu,§

George M. Yousef,∥ and Sergey N. Krylov*,†

†Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3,Canada‡Sunnybrook Research Institute and Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University ofToronto, Toronto, Ontario M5S 1A8, Canada§Department of Radiation Oncology, Sunnybrook-Odette Cancer Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5,Canada∥Keenan Research Centre, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada

*S Supporting Information

ABSTRACT: Direct quantitative analysis of multiple miRNAs (DQAMmiR) is ahybridization-based assay, in which the excess of the DNA hybridization probes isseparated from the miRNA-probe hybrids, and the hybrids are separated from eachother in gel-free capillary electrophoresis (CE) using two types of mobility shifters:single-strand DNA binding protein (SSB) added to the CE running buffer andpeptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNAhybridization probes and requires no SSB in the CE running buffer. PNA probes areelectrically neutral, while PNA−miRNA hybrids are negatively charged, and thisdifference in charge can be a basis for separation of the hybrids from the probes. Inthis proof-of-principle work, we first experimentally confirmed that the PNA−RNAhybrid was separable from the excess of the PNA probe without SSB in the runningbuffer, resulting in a near 10 min time window, which would allow, theoretically,separation of up to 30 hybrids. Then, we adapted to PNA−RNA hybrids ourpreviously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modifiedtheoretical model indicated that PNA−RNA hybrids of slightly different lengths could be separated from each other withoutdrag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids ofdifferent lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental modelincluded three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes hadlengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; theother two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrationssuggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.

MicroRNAs (miRNAs) are short single-strand RNAmolecules (18−25 nucleotides) that play key roles in

fundamental cellular processes such as cell differentiation,proliferation, and apoptosis.1,2 Aberrant miRNA expressionaffects these processes and, as a result, leads to variouspathological outcomes. In particular, abnormal miRNAexpression has been linked with Alzheimer’s disease, heartdiseases, and multiple cancers.3−5 For example, abnormalexpression of groups of 2, 3, and 4 miRNAs were found to beindicative of prostate, lung, and breast cancers in humans.6−8

Clinical use of reliable miRNA-based disease biomarkersrequires accurate and robust quantitative analyses of multiplemiRNAs in clinical samples.

Many existing methods of miRNA detection, includingmicroarray-based techniques, quantitative reverse transcriptionpolymerization chain reaction, and next-generation sequencing,are indirect, i.e., they require modification or amplification ofmiRNA targets. Such manipulations with the targets introducesequence-related biases in the analyses.9,10 Other techniqueshave also been developed for direct quantitation of multiplemiRNAs, such as mass spectrometry,11,12 electrochemicalsensing,13,14 optical resonance,15 and etc.16−19 However,methods based on these techniques often require isolation of

Received: October 18, 2018Accepted: November 19, 2018Published: November 19, 2018

Article

pubs.acs.org/acCite This: Anal. Chem. 2018, 90, 14610−14615

© 2018 American Chemical Society 14610 DOI: 10.1021/acs.analchem.8b04793Anal. Chem. 2018, 90, 14610−14615

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total RNA from the sample before measuring miRNA; suchsample processing involves a time-consuming purification stepand introduces analytical inaccuracies.20,21 There are very fewmethods that can satisfy the criteria of direct quantitation ofmultiple miRNAs with minimal sample processing.13,17 One ofsuch approaches is direct quantitative analysis of multiplemiRNAs (DQAMmiR) by gel-free capillary electrophoresis(CE),22 which has been proven to be robust in analyzingmiRNAs in crude cell lysate.23 In general, DQAMmiR is ahybridization-based assay, in which the excess of hybridizationprobes (fluorescently labeled for sensitive detection) isseparated from the miRNA-probe hybrids, and the hybridsare separated from each other, by CE with strong electro-osmotic flow (EOF). The EOF propels the probes and thehybrids toward the detector at the end of the capillary;quantitative detection of the probes and the hybrids facilitatesabsolute quantitation of the hybrids without calibration curves.The first generation of DQAMmiR utilizes ssDNA probes

and two types of mobility shifters: (i) ssDNA binding protein(SSB) added to the running buffer24 and (ii) different-length-peptide drag tags conjugated onto the ssDNA probes.25 SSBfacilitates separation of the excess probes from the hybrids bybinding the ssDNA probes but not the hybrids. The drag tagsinduce mobility shifts in the hybrids, allowing their separationfrom each other without interference with the binding affinityof SSB to the ssDNA probes. However, SSB is a protein, andwhile it is quite stable, involving it in the assay can potentiallydecrease the assay’s robustness and increase its cost, making itless suitable for practical clinical use.To address this issue, here, we introduce the second-

generation DQAMmiR, which does not need SSB and haslesser dependence on drag tags on the probes. The function ofSSB, utilized in the first-generation DQAMmiR, is to separatethe excess ssDNA probes from the ssDNA−miRNA hybrids,which have similar electrophoretic mobilities due to negativecharges on both DNA and RNA. SSB would not be needed ifthe probes were polymers capable of hybridizing miRNA butnot being negatively charged. An example of such polymers ispeptide nucleic acid (PNA), which is an electrically neutralanalogue of DNA composed of repeating N-(2-amino-ethyl)-glycine units.26 PNA−miRNA hybrids are negatively chargeddue to the negative charge on miRNA, although the chargedensity is about half of that of DNA−miRNA hybrids.Electrically neutral PNA probes should be separable from thenegatively charged PNA−miRNA hybrids without any addi-tional mobility modifier, such as SSB.To achieve quantitation of multiple targets, separating the

hybrids from each other is also essential. A theoretical modelwas previously developed to predict the electrophoreticmobilities of DNA−RNA hybrids of the first-generationDQAMmiR.27 Here we adapted this model to PNA−RNAhybrids of the second-generation DQAMmiR. The theoreticalresults indicated that the PNA−RNA hybrids with differentnumbers of base pairs could be separated from each otherwithout drag tags, while the peptide drag tags on the PNAprobes would still be useful mobility shifters for PNA−RNAhybrids of the same length. The proposed model suggestedthat using PNA probes in DQAMmiR would simplify the assayvia two ways: by making SSB unnecessary and by decreasingthe number of probes conjugated with drag tags. The basicschematic of the second-generation DQAMmiR with PNAhybridization probes is shown in Figure 1. We proved thefeasibility of PNA-facilitated DQAMmiR by analyzing three

miRNAs (miR-21, miR147a, and miR-378g), which have 20,20, and 22 nucleotides, respectively. High accuracy andprecision along with low LOQ indicate that DQAMmiR withPNA hybridization probes may potentially serve as a tool forpractical miRNA analyses in clinical samples.

■ MATERIALS AND METHODSMiRNAs and PNA Probes. All miRNAs were custom-

synthesized by IDT (Coralville, IA, USA). All PNA probeswere custom-synthesized by PNA Bio (Thousand Oak, CA,USA). Detailed information on miRNAs and PNA probes canbe found in the Supporting Information. Concentrations ofmiRNA and PNA probes in stock solutions were determinedusing light absorbance at 260 nm measured with a NanoDropND-1000 spectrophotometer (Thermo-Fisher Scientific, Wal-tham, MA, USA); the extinction coefficients were provided byrespective suppliers of miRNA and PNA. Acetonitrile (20%, v/v) was added to all buffers to improve PNA solubility.

Hybridization Reactions. Hybridization was carried outin a Mastercycler 5332 thermocycler (Eppendorf, Hamburg,Germany). Various concentrations of three miRNAs wereincubated with 10 nM of their respective PNA probes in theCE running buffer (20 mM Borax, 120 mM NaCl, 20%acetonitrile, pH 9.0). According to the information providedby the supplier of PNA, all the PNA−miRNA hybrids used inthis study have a melting temperature (Tm) above 80 °C. Thus,we selected 60 °C as the hybridization temperature, which isabout 20 °C lower than their Tm and favorable for thehybridization reactions.28 The temperature was first increasedto a denaturing level of 95 °C and then lowered to 60 °C at arate of 20 °C/min. After that, the temperature was held at 60°C for 30 min to allow hybridization. To minimize RNAdegradation, a nuclease-free environment was maintained whilehandling miRNA samples.

Capillary Electrophoresis. All experiments were per-formed using a P/ACE MDQ CE instrument (SCIEX, Brea,CA, USA) equipped with a laser-induced fluorescencedetector. We used bare fused-silica capillaries with an outerdiameter of 365 μm, an inner diameter of 50 μm, and a totallength of 80 cm. The distance from the injection end of the

Figure 1. Schematic depiction of DQAMmiR with PNA hybridizationprobes.

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capillary to the detector was 70 cm. The capillary was flushedprior to every CE run with methanol, 0.1 M HCl, 0.3 MNaOH, deionized H2O, and the running buffer for 1 min eachunder a 20 psi pressure. The sample was injected into thecapillary by a 5 s pressure pulse of 0.5 psi. Electrophoresis wasdriven by an electric field of 312.5 V/cm with positive polarityat the capillary inlet. The capillary coolant temperature waskept at 20 °C during CE experiments. Fluorescence of theAlexa488 label on the PNA probes was excited by 488 nm lightgenerated with a continuous-wave solid state laser (JDSU,Milpitas, CA, USA). Electropherograms were analyzed with 32Karat Software. Peak areas were divided by the correspondingmigration times to compensate for the dependence of theresidence time in the detector on the electrophoretic velocityof the analyte. The quantity of each miRNA was calculatedusing these peak areas (see the Supporting Information fordetails).

■ RESULTS AND DISCUSSIONIn DQAMmiR, accurate quantitation of multiple miRNAsrelies on the separation of the nonhybridized probes from themiRNA-probe hybrids and hybrids from each other by CE. Toestablish DQAMmiR with PNA probes, it is necessary toinvestigate the separation of the PNA probe from the PNA−miRNA hybrid. A proof-of-principle experiment was performedusing miR-21 and its complementary PNA probe. The resultconfirmed that the negatively charged PNA−RNA hybrid wasseparable from the excess of the neutral PNA probe withoutany mobility shifter (Figure 2). A near 10 min time window

was obtained between the PNA probe and the PNA−miRNAhybrid, which would allow theoretical separation of up to 30hybrids (Supporting Information). The observed quality ofseparation of the excess PNA probe from the hybrid suggeststhat this PNA-facilitated assay could analyze miRNAs withhigh multiplexing capacity.Another essential step for PNA-facilitated DQAMmiR was

to separate PNA−miRNA hybrids from each other. For thispurpose, theoretical estimates of the electrophoretic mobilityof PNA−RNA hybrids were found by adapting to thesehybrids a theoretical model previously developed for DNA−RNA hybrids.27 In that model, the DNA−RNA duplex wasassumed to behave like a rigid rod, because the contour lengthsLhyb = Nhybb (Nhyb is the number of base pairs, and b is thehelical rise per base) for such short hybrids (18−25 bp) areshorter than their Kuhn length bK. For the case of PNA−RNA

duplexes, b = 0.24 nm leads to 4.32 nm ≤ Lhyb ≤ 6 nm (since18 ≤ Nhyb ≤ 25),29 while the Kuhn length of these helices canbe estimated to be larger than 60 nm (bK > 60 nm)(Supporting Information). Thus, the condition of Lhyb << bK isalso satisfied in the case of PNA−RNA duplexes, and the rigid-rod model can be adapted to predict the mobilities of PNA−RNA hybrids. Thus, the expressions for the electric force(FE.hyb) and hydrodynamic friction force (Ff,hyb) are

30,31

FeL

zE

iE,hyb

hyb

Bλ=

(1)

i

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y

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L

d2 ln

20.72f,hyb hyb rel

hyb

hyb

1

πη= −−

(2)

Here Lhyb = Nhybb is the length of the hybrid, and dhyb is thediameter of the hybrid, which is approximately 3.5 nm forPNA−RNA duplexes.29 zi is the valence of the counterions. λBand η are the Bjerrum length and viscosity of the solution,which can be found to be 0.79 nm and 1.10 mPa·s,respectively, for the 20% (v/v) acetonitrile-containing aqueoussolutions at 20 °C (Supporting Information). urel is the velocityof the hybrid with respect to the running buffer. Thus, themobility of the PNA hybrid can be found from the balance ofthe electric force and the hydrodynamic friction force exertedon it.

F FE,hyb f,hyb= (3)

Combining eqs 1−3, the expression for electrophoreticmobility μhyb can be written as

i

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ez

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hyb

hybμ

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(4)

Eq 4 suggests that the mobility of a PNA−RNA hybriddepends on the number of base pairs of the hybrid (Nhyb).Such a dependence was also found in our previous model ofthe DNA−RNA hybrids, although we neglected this effect andassumed for simplicity that different-length DNA−RNAhybrids had the same mobility.27 This dependence is strongerfor PNA−RNA hybrids because of the smaller value of b/dhybfor the PNA−RNA helix (b = 0.24 nm, dhyb = 3.5 nm) ascompared with the DNA−RNA helix (b = 0.34 nm, dhyb = 2.6nm) (See Figure S1 in the Supporting Information).The validity of this model was tested by theoretically

estimating the electrophoretic mobility of PNA-miR-21 (Nhyb= 22) and comparing it with the experimentally measuredvalue. The electrophoretic mobility of the PNA-miR-21 hybridwas calculated to be 1.12 × 10−4 cm2·V−1·s−1, while theexperimental result led to a value of 1.20 × 10−4 cm2·V−1·s−1

(as shown in Figure 2). The small deviation of the calculatedvalue from the experimental value suggested the model’sapplicability to PNA−RNA hybrids. Simulated electrophoro-grams of PNA−RNA hybrids with different numbers of basepairs were obtained by using μEOF = 3.14 × 10−4 cm2·V−1·s−1

(which corresponds to our running buffer containing 20 mMBorax and 20% acetonitrile at pH 9.0) as shown in Figure 3A.It is clear that a single-base-pair difference in lengths of PNA−RNA hybrids can cause significant difference in theirelectrophoretic mobilities. Such difference in mobilities shouldallow the separation of different-length PNA−miRNA hybridsfrom each other.

Figure 2. CE separation of a PNA probe from a PNA-miR-21 hybridin a running buffer of 20 mM Borax and 20% acetonitrile at pH 9.0.The sample was prepared by incubating 2 nM miR-21 with a 10 nMPNA probe with a sequence fully complementary to miR-21.Separation was driven by an electric field of 312.5 V/cm at 20 °C.

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On the other hand, molecules with similar mobility, such asdifferent-sequence but same-length PNA−miRNA hybrids, canalso be electrophoretically separated from each other by end-labeled free solution electrophoresis (ELFSE).32 By using thisapproach, we have developed universal peptide drag tagsconjugated on the ssDNA probes as mobility shifters toseparate ssDNA−miRNA hybrids in our first-generationDQAMmiR.25 In this case, these peptide drag tags could alsobe applied to facilitate separation of different-sequence butsame-length PNA−miRNA hybrids. For this purpose, we alsoestimated the electrophoretic mobility of PNA−RNA hybridsconjugated with different-length peptide drag tags. When thehydrodynamic friction force acting on the drag tag (Ff,tag) istaken into account, eq 3 can be rewritten as

F F FE,hyb f,hyb f,tag= + (5)

The hydrodynamic force, Ff,tag, acting upon the drag tag canbe described via the expression

F R u6f,tag H,tag relπη= (6)

where RH,tag is the hydrodynamic radius of the drag tag. Bysubstituting eqs 1, 2, and 6 for FE,hyb, Ff,hyb, and Ff,tag,respectively, into 5 and by solving the resulting equation, wecan obtain the expression for electrophoretic mobility ofPNA−RNA hybrids with peptide drag tags

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Studies of unfolded peptides have resulted in the followingdependence for the hydrodynamic radius RH,tag (in nm) ofshort peptides by fitting empirical data33

R N(0.22 0.11)H,tag tag0.57 0.02= ± ±

(8)

Here, Ntag is the number of amino-acid residues in the peptidechain. The hydrodynamic radii of peptide drag tags withdifferent numbers of amino-acid residues can be estimatedusing eq 8. Thus, mobilities of PNA−RNA hybrids withdifferent-length peptide drag tags could be estimated. As anexample, the simulated electropherograms of 20-bp PNA−

RNA hybrids with different-length peptide drag tagsconjugated on the probe are shown in Figure 3B. Theestimated electrophoretic mobilities of different PNA−miRNAhybrids can be found in the Supporting Information, indicatingthat the PNA−miRNA hybrids could be separated from eachother by either the difference in numbers of nucleotides orlengths of peptide drag tags. The simulation strongly suggeststhat using PNA probes can simplify DQAMmiR by reducingthe need for conjugation of drag tags.As proof-of-principle, three miRNA targets, miR-147a, miR-

378g, and miR-21, which have the numbers of nucleotides of20, 20, and 22, respectively, were analyzed by DQAMmiR withPNA hybridization probes. Two PNA probes complementaryto miR-378g and miR-21 were custom-synthesized withoutdrag tags, while a PNA probe complementary to miR-147a wasconjugated with a five-amino-acid tag.We first performed CE separation with a running buffer of

20 mM Borax and 20% acetonitrile at pH 9.0 (Figure 4A). As a

result, the 22-bp PNA−miRNA-21 hybrid exhibited thehighest electrophoretic mobility, while the PNA-miR-147a(20 bp, 5 aa) was found to have the lowest electrophoreticmobility, which agreed well with our theoretical predictions.While deviations of 29, 24, and 6% were found between thetheoretically predicted mobilities of PNA-miR-147a (20 bp, 5aa), PNA-miR-378g (20 bp), and PNA-miR-21 (22 bp) andtheir respective experimentally measured mobilities, thequalitative agreement between the theoretical prediction andexperimental results was evident: (i) the electrophoreticmobility of the PNA−miRNA hybrid depended on the numberof base pairs in the hybrid, and the shorter hybrid had thelower electrophoretic mobility; (ii) the mobility of the hybridcould be altered by conjugating with a peptide drag tag on thePNA probe. However, a mobility shift by the 5-aa peptide tagon the PNA-miR-147a hybrid was not sufficient for baselineseparation of the two 20-bp hybrids (baseline separation ispreferable for high accuracy of miRNA quantitation). Theresolution in CE separation can be improved by suppressingthe EOF of the running buffer,34 which can be achieved, inparticular, by increasing the ionic strength of CE runningbuffer. Here, we added 120 mM NaCl into the running buffer

Figure 3. Simulated electropherograms of different PNA−RNAhybrids. (A) PNA−RNA hybrids with numbers of base pairs (Nhyb) ina range of 18−25. (B) PNA−RNA hybrids (20 bp) with lengths(numbers of amino acids) of peptide tags (Ntag) in a range of 0−30.Migration times of peaks shown above were estimated by using μEOF =3.14 × 10−4 cm2·V−1·s−1, which was experimentally measured for ourCE running buffer containing 20 mM Borax and 20% (v/v)acetonitrile at pH 9.0.

Figure 4. (A) Comparison of experimental and predicted separationsof three PNA−miRNA hybrids in 20 mM Borax and 20% acetonitrileat pH 9.0. (B) Baseline separation of the three hybrids from eachother by using a high-ionic-strength CE running buffer: 20 mM Borax,120 mM NaCl, 20% acetonitrile, pH 9.0. Peak assignment: (a) excessPNA probes, (b) PNA-miR-147a (20 bp, 5 aa), (c) PNA-miR-378g(20 bp), and (d) PNA-miR-21 (22 bp).

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to suppress EOF and prolong separation. As a result, baselineseparation was achieved, leading to a peak resolution of 3.1between PNA-miR-147a and PNA-miR-378g (Figure 4B).Thus, the running buffer for the following experiments was 20mM Borax, 120 mM NaCl and 20% acetonitrile at pH 9.0.To study the suitability of PNA-facilitated DQAMmiR for

miRNA quantitation, measurements of miR-147a, miR-378g,and miR-21 were performed simultaneously in a concentrationrange of 32−500 pM, which is of interest for analyzingmiRNAs in clinical samples.22 In all experiments, finalconcentrations of all three PNA probes in hybridizationmixtures were set at 10 nM, which is an excess to all targets.The electropherograms obtained in these experiments areshown in Figure 5A. Concentrations of individual miRNAs

were calculated by using a math strategy developed in theprevious DQAMmiR study (Supporting Information)22

Aq q

q

AmiRNA

( P )ii

i iiN i i

iN A

q

H

H P

1 P 0

P 1

i

iH

H

[ ] =∑ [ ]

+ ∑=

= (9)

[P]0i is the total concentration of the i-th PNA probe

(composed of the hybrid and the excess probe), AHi is the area

corresponding to the i-th hybrid, AP is the cumulative area ofthe unbound probes, qP

i is a relative quantum yield of the i-thPNA probe to normalize the quantum yield differencesbetween the probes, and qH

i is the relative quantum yield ofthe i-th hybrid with respect to that of the unbound probe. BothqPi and qH

i were determined in separate experiments(Supporting Information). Using eq 9, we were able todetermine concentrations of the three targets simultaneouslyby quantitating peak areas from a single electropherogramwithout building/using a calibration curve.The measured concentrations of the three targets were

compared with their actual concentrations as shown in Figure5B. A recovery of over 90% with RSD of approximately 10%was observed for measuring all three miRNAs with

concentrations varying in a 32−500 pM range (SupportingInformation). Also, by studying the signal-to-noise ratio (SNR)of the peaks of hybrids, the lower limit-of-quantitation (LOQ)of this method performed with a commercial CE-LIFinstrument was determined to be 14 pM. These resultsdemonstrate that this PNA-facilitated DQAMmiR is capable ofquantitating multiple miRNAs with high accuracy andprecision at picomolar concentrations.In contrast to DNA probes, PNA probes have greater

specificity in hybridizing with DNA or RNA, with a PNA−DNA base mismatch being more destabilizing than a similarmismatch in a DNA−DNA duplex.35 This property will allowus to establish PNA-facilitated DQAMmiR for analyzingmiRNAs with slightly different sequences by simply changingconditions of the hybridization reaction, such as temperature,addition of denaturing reagent, and etc. PNAs are also resistantto degradation by nucleases or proteases,36 making themperfect hybridization probes for analysis of miRNAs inbiological samples. As a result, PNA-facilitated DQAMmiRcan be potentially a practical tool for quantitation of miRNAsin clinical samples.

■ CONCLUSIONSIn this study, we introduced the second-generation DQAM-miR, which utilizes electrically neutral PNA probes instead ofnegatively charged DNA probes. In this approach, thenegatively charged PNA−miRNA hybrids were separatedfrom the neutral PNA probes by CE without any additionalmobility shifter, leading to a near 10 min time window, whichwould allow us to analyze up to 30 miRNAs simultaneously.The theoretical study of the electrophoretic mobility of PNA−miRNA hybrid qualitatively predicted that the separation ofthe hybrids from each other could be achieved by utilizingtheir differences in either (i) the number of base pairs or (ii)the length of peptide drag tags. This was confirmed by ourexperimental results. Our proof-of-principle study demonstra-ted that PNA-facilitated DQAMmiR was able to quantify threemiRNAs simultaneously with high accuracy (recovery > 90%)and precision (RSD ≈ 10%) with a LOQ of 14 pM by using acommercial CE instrument, suggesting that DQAMmiR withPNA hybridization probes may facilitate validation and clinicaluse of miRNA biomarkers.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.8b04793.

Sequences of miRNA targets and their PNA probes;estimation of the multiplexing capacity in the separationwindow; assumption of the short PNA−miRNA hybridbehaving as a rigid rod; the Bjerrum length (λB) andviscosity (η) of the 20% (v/v) containing solutions;dependence of the relative mobility of a hybrid on thenumber of base pairs of the hybrid; theoretical estimatedelectrophoretic mobilities of PNA−miRNA hybrids;simulated electropherograms of the PNA−miRNAhybrids; relative quantum yield measurements; deriva-tion of equations for the determination of concen-trations of multiple miRNA by PNA-facilitated DQAM-miR; quantitation results of PNA-facilitated DQAMmiR(PDF)

Figure 5. (A) Electropherograms of PNA-facilitated measurements.Peak assignment: (a) excess PNA probes, (b) PNA-miR-147a (20 bp,5 aa), (c) PNA-miR-378g (20 bp), (d) PNA-miR-21 (22 bp).Electropherograms i−vi correspond to target concentrations of 500,250, 125, 63, and 32 pM, respectively. B) Quantitation of threemiRNAs simultaneously by PNA-facilitated DQAMmiR. Concen-trations measured with DQAMmiR are shown with respect to theiractual values determined by light absorbance at 260 nm. miR-21, miR-147a, and miR-378g are represented by black rectangles, red circles,and blue triangles, respectively. The dashed line (y = x) represents aline corresponding to 100% recovery. Error bars show one standarddeviation from mean values obtained from three experiments.

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■ AUTHOR INFORMATIONCorresponding Author*E-mail: skrylov@yorku.ca.ORCIDLiang Hu: 0000-0003-4507-8525Svetlana M. Krylova: 0000-0002-3291-6721Burton B. Yang: 0000-0002-2892-7209Stanley K. Liu: 0000-0001-6851-0857George M. Yousef: 0000-0003-3859-779XSergey N. Krylov: 0000-0003-3270-2130Author ContributionsLiang Hu and Mansi Anand contributed equally to thismanuscript. The manuscript was written through contributionsof all authors. All authors have given approval to the finalversion of the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Canadian Institutes of Health Research (grantnumber CPG 134747) and the Natural Sciences andEngineering Research Council of Canada (grant numberCHRP 462341-2014) for financial support.

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Analytical Chemistry Article

DOI: 10.1021/acs.analchem.8b04793Anal. Chem. 2018, 90, 14610−14615

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