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Expert Review of Molecular Diagnostics

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What is the potential of nanolock– andnanocross–nanopore technology in cancerdiagnosis?

Li-Qun Gu, Kent S. Gates, Michael X. Wang & Guangfu Li

To cite this article: Li-Qun Gu, Kent S. Gates, Michael X. Wang & Guangfu Li (2018) What is thepotential of nanolock– and nanocross–nanopore technology in cancer diagnosis?, Expert Review ofMolecular Diagnostics, 18:2, 113-117, DOI: 10.1080/14737159.2018.1410060

To link to this article: https://doi.org/10.1080/14737159.2018.1410060

Accepted author version posted online: 24Nov 2017.Published online: 01 Dec 2017.

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EDITORIAL

What is the potential of nanolock– and nanocross–nanopore technology in cancerdiagnosis?Li-Qun Gu a, Kent S. Gates b, Michael X. Wangc and Guangfu Lid

aDepartment of Bioengineering and Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA; bDepartment of Chemistryand Department of Biochemistry, University of Missouri, Columbia, MO, USA; cDepartment of Pathology and Immunology, Washington UniversitySchool of Medicine, St. Louis, MO, USA; dDepartment of Surgery and Ellis Fischel Cancer Center, University of Missouri, Columbia, MO, USA

ARTICLE HISTORY Received 4 October 2017; Accepted 23 November 2017

KEYWORDS Cancer diagnostics; nanopore; nanolock; nanocross; biosensor; driver mutation; single nucleotide polymorphism (SNP); Point-of-care; precisiononcology; Precision medicine

1. Driver mutations, cancer, and detection methods

Mutation is the permanent change in the DNA sequence of agenome. Mutations can alter the function of encoded proteinswith pathophysiological consequences. Driver mutations are atype of gene alteration that can cause dysregulated cell divi-sion and give a growth advantage, ultimately leading to tumordevelopment and progression [1,2]. Driver mutations are ahallmark of cancer. For example, mutations in BRCA1/2genes are closely correlated with breast cancer risk [3]; lungcancers bearing mutated EGFR respond to EGFR inhibitors[4,5]; and melanomas bearing mutated BRAF usually respondto BRAF inhibitors [6,7]. As driver mutations are cancer bio-markers, they have been targeted in precision oncology, forcancer diagnosis, tailored therapy, and treatment responsemonitoring [8,9].

Molecular cancer diagnostics requires sensitive detection ofdriver mutations at a very low frequency in early stages ofcancer, and accurate quantification of mutation frequency in acomplex biological sample [10]. Although many methods havebeen developed for the detection of mutation [11,12], thereare still limitations in terms of clinically availability, sensitivity,specificity, and efficiency. Currently, PCR-based amplificationmethods including allele-specific PCR and digital PCR arewidely used to detect cancer gene mutation in the clinicsetting. These technologies require expensive instrumenta-tion, reagents, well-trained personnel and standardizedlaboratory protocols, and the results may be compromisedby contamination [13,14]. The next generation sequencing(NGS) technology is a most promising method for highthroughput cancer genome profiling and residual diseasedetection. However, its inherent weakness such as long turn-around-time (usually several weeks), high cost, requirement ofadequate tissue source of nucleic acids, sequencing librarypreparation, complex bioinformatics, currently hinders its suc-cessful implementation in clinical practice [15–17].Development of novel strategies for the accurate detectionof gene mutations, in particular point mutations (single-basechanges) or single nucleotide polymorphism (SNP), is urgentlyneeded [18–20].

In this review, we describe two different single-moleculeplatforms, nanolock-nanopore [21] (Figure 1(a)) and nano-cross–nanopore [22,23] biosensers (Figure 1(b)) for the detec-tion of point mutations. The test-bed of these studies is theBRAF V600E mutation, which occurs with high prevalence inseveral types of cancer and is the target of several drugs [24].Accurate detection of this SNP could help physicians diagnosecancers earlier and treat them with individualized therapies.Using the two nanopore methods, we are able to directly‘read’ single DNA molecules of the low frequency BRAFV600E allele against a background of the wild-type sequence,and quantify the mutation frequency in tumor tissues of can-cer patients.

2. Nanolock–nanopore and nanocross–nanoporesensors for SNP detections

The sensing element is a nanopore, a 2-nm-wide proteinchannel that is provided by α-hemolysin embedded in a lipidmembrane. A transmembrane voltage is applied to drive anion current through the pore. When a target molecule in thesolution passes through the nanopore, it changes the currentin a characteristic manner, thus producing an electric signa-ture that enables single molecule target detection. In theBRAF V600E detection, we used a DNA probe that can hybri-dize with both mutant and wild-type alleles to form themutant•probe and wild-type•probe complexes. Powered bythe electrophoretic potential of the device, both complexescan be captured by the nanopore and subsequently unzippedby this molecular machine. In order to selectively discriminatethe mutant BRAF allele, we have constructed the mutationsequence-specific nanolock and nanocross motifs.

Nanolock is a class of inter-strand stabilizing chemical struc-tures that is formed by binding of a ligand [25–28] at specificbase pairs. Fundamentally the nanolock exploits the concept ofa stimuli-responsive nucleic acid [29], in which the polymer’sproperties are altered in response to an environmental stimulussuch as ligand binding. For detection of BRAF V600E [21](Figure 1(a)), we designed a special probe that forms

CONTACT Li-Qun Gu gul@missouri.edu; Kent S. Gates gatesk@missouri.edu

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2018VOL. 18, NO. 2, 113–117https://doi.org/10.1080/14737159.2018.1410060

© 2017 Informa UK Limited, trading as Taylor & Francis Group

the thymine–thymine (T–T) mismatched base pair in themutant•probe complex. The T–T mismatch can be bound witha mercuric ion (Hg2+) to form a stabilizing T–Hg2+–T ‘nanolock’[25,30–33]. In contrast, the wild-type•probe complex does notcontain the T–T mismatch and thus cannot form the nanolock.Most importantly, due to the high stability of the nanolock, themutant•probe unzipping process in the nanopore splits intotwo consecutive steps that can be resolved in the nanopore.In contrast, the unzipping of the wild-type•probe complex with-out the nanolock remains a transient, cooperative, one-stepprocess. This mechanistic difference in the unzipping processesallows the locked mutant•probe complex to generate a distinct

nanopore signature. The nanolock-produced unzipping signa-ture functions as an ‘electrical fingerprint’, enabling discrimina-tion of the mutant allele from the wild-type sequence. With thefingerprint, we are not only able to ‘read’ each mutant allelemolecules, but also quantify the mutation frequency (mutationpercentage) in a tumor tissue sample by measuring the occur-rence of the nanolock signature.

We have also developed a separate approach that exploitssequence specific cross-linking with a reactive probe strandthat covalently captures the mutant BRAF target sequence[22,23] (Figure 1(b)). In this ‘nanocross’ approach, a DNA abasicsite, embedded in a specialized probe sequence, enables

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Figure 1. Single-molecule detection of cancer driver mutation by using nanolock-nanapore and nanocross-nanopore sensors. (a) Nanolock-nanopore detection ofthe BRAF V600E mutant allele in tumor tissue DNA sample; (b) Nanocross-nanopore detection of the BRAF V600E mutant DNA sequence. In both methods, the non-covalent nanolock (a) or covalent nanocross (b) is constructed only on the mutant•probe DNA complex. Their special molecular configuration changes in thenanopore produce a unique ion current signature (current blockade marked by red solid triangle), which serves as a fingerprint of a single mutant DNA molecule.For the nanocross method (b), the unique long-duration blockade by the nanocross enables multi-nanopore simultaneous detection of mutant DNA;(c) Identification of mutant fingerprints enables Yes/No early cancer diagnostics, and counting of mutant fingerprints allows quantitation of mutant frequencyfor personalized treatment. This figure was adapted with permission from (Wang Y, Tian K, Shi R et al. Nanolock-Nanopore Facilitated Digital Diagnostics of CancerDriver Mutation in Tumor Tissue. ACS sensors, 2(7), 975–981 (2017)). Copyright (2017) American Chemical Society; adapted with permission from (Zhang X, Price NE,Fang X, Yang Z, Gu LQ, Gates KS. Characterization of Interstrand DNA-DNA Cross-Links Using the alpha-Hemolysin Protein Nanopore. ACS nano, 9(12), 11812–11819(2015)). Copyright (2015) American Chemical Society; adapted from (Nejad MI, Shi R, Zhang X, Gu LQ, Gates KS. Sequence-Specific Covalent Capture Coupled withHigh-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence. ChemBioChem, 18(14), 1383–1386 (2017)) with permission of John Wiley and Sons,Copyright (2017).

114 L. GU ET AL.

highly selective covalent cross-linking to adenine 1799 in theBRAF V600E mutant gene sequence [34]. Importantly, thecross-linked probe–target complex gives an unmistakable,long-lasting current block in the nanopore device [22,23].This current signature is easily distinguished from the short-duration blocks caused by uncross-linked target•probe com-plexes derived from the wild-type sequence. Accordingly, weshowed that the fraction of T > A mutant sequence could bequantitatively determined within mixtures containing a highbackground of the wild-type allele [23].

3 Advantages, improvement, and applications

The nanopore has been broadly investigated for variousgenetic [35–39], epigenetic [40–43] and proteomic [44–46]detection strategies [47]. Many excellent studies have demon-strated nanopore’s single-nucleotide sensitivity [48–50], andability to detect single-nucleotide polymorphism [49,51–53].However, their applications in point mutation detection inclinical setting have not been explored. The nanopore-basednext-generation sequencing technology is being developed[52,54–57], but currently the sequencing accuracy (90%)remains impracticable for detecting cancer mutations at lowfrequencies [58]. By comparison, a great advantage of thenanolock–nanopore and nanocross–nanopore sensors is theirhigh sensitivity. Due to the capability to discriminate eachindividual mutation DNA molecules captured by the nanopore,the sensor in principle can detect mutant alleles at very lowfrequency. For BRAF V600E, the sensor has been used to identifymutant alleles at 1% in the presence of wild-type DNA. Thissensitivity, which can be further enhanced upon systemimprovement (see later), is superior to commercially availablemutation detectionmethods such as polymerase chain reaction(PCR) and next generation sequencing (NGS) [21]. In addition tohigh sensitivity, both nanolock– and nanocross–nanopore sen-sors exhibit their own advantages. The non-covalent nanolockcan be formed immediately upon addition of the ligand in thesolution, therefore vastly decreasing the time for sample pre-paration prior to detection. By comparison, the formation of thecovalent nanocross takes much longer time. However, theresulting long-lasting blockade is a unique signature for unmis-takable discrimination of the mutant DNA. The nanocross sig-nature can be easily terminated by reversing the voltagepolarity (to release the cross-linked DNA duplex into the solu-tion) to capture the next DNA duplex. In addition, due to thenanocross’ long block duration, we can record the time-depen-dent capture of cross-linked duplexes in a device with multiplenanopores embedded in the membrane. The current trace forsuch an experiment is characterized by stepwise current dropscorresponding to sequential capture of cross-linked duplexesfrom the test solution (Figure 1(b), right current trace). Becausethe single-molecule capture rate is proportional to the concen-tration of cross-links in the cis solution and the number ofnanopores in the membrane, the multi-channel method willnot only vastly shorten the detection time, but also increase thesensitivity, making it useful in particular for detection at lowmutant frequency.

To enable future clinical applications, we need improvethe performance of both nanopore sensors, including

applicability, throughput and sensitivity. First, the nanolockand nanocross toolbox must be expanded in order to targeta broader spectrum of driver mutations in human cancers(e.g. mutations on KRAS and EGFR). Among various SNPs, T orA can be detected using the T–Hg2+–T nanolock and nano-cross; C or G can be detected using the C–Ag+–C nanolock[25,30–33,43]. In addition, many mismatch-specific chelatingcompounds [26,27] and metallointercalators [28] with greatbase-pair selectivity can be utilized to construct mutation-targeted nanolocks. Similarly, additional cross-linking motifswill be developed for the covalent capture of various targetsequences. Furthermore, because accurate clinical diagnos-tics requires simultaneous detection of multiple mutationsrather than just one, we will develop methods such as ‘nano-pore barcoding’ (exploiting a method we developed for mul-tiple microRNA detection [42]) for simultaneous detection ofmultiple biomarker mutations. Last, the analytical procedurein the current stage could be slow (low DNA capture rate)due to the low DNA concentrations. To enhance the detec-tion efficiency, one solution is utilizing miniaturized nano-pore system such as droplet nanopore [45] and nanoporechips [58]. The miniaturized device allows using very smallvolume (1 µl) of solution. Therefore the detection can beperformed in pre-concentrated and enriched sample withhighly elevated single molecule capture rate. Ultimately, itmay be possible to realize amplification-free detection in aclinical setting.

Early cancer diagnostics demand detection of mutations atvery low frequency. The nanocross– and nanolock–nanoporesensors ultimately may be sufficiently sensitive to execute thistask (Figure 1(c)). Since this single-molecule detection technol-ogy can determine if a DNAmolecule is a mutant or wild-type, itsfalse-positive and false-negative rates are both low. Ideally,observation of just one mutant DNA in a given time (e.g. 1 h)would immediately suggests the presence of that specific muta-tion in the sample. Longer detection time results in highersensitivity, because the chance to capture a single mutant DNAmolecule increases as the detection time increases. Therefore,the techniques, in essence, yield digital signals that are sensitiveenough to perform ‘yes/no binary diagnostics’ of a cancer muta-tion, allowing doctors to quickly take appropriate clinical action.

Recent studies have demonstrated that circulating tumorDNA (ctDNA) fragments carry tumor specific sequence altera-tions even in the early stages of cancer [59]. Present in thecell-free fraction of blood ‘liquid biopsy’, ctDNA offers a non-invasive ‘real-time’ biomarker of the disease [59–61]. Whileclinical use of nanolock– and nanocross–nanopore sensorsfor ctDNA detection remains to be explored, the initial resultsoffer a possibility for a new non-invasive way to assist cancerearly diagnostics, potentially leading to improved patient sur-vival and quality of life. The promise of commercially availableportable nanopore devices means that this approach couldfacilitate the early diagnosis of disease in low-resource clinicalenvironments.

Because these nanopore techniques can be used to accuratelyquantify the frequency of a particular mutation (Figure 1(c)), theclinical investigator would be able to use it for monitoring thelevels of cancermutations in patients being treatedwith particulardrugs or other interventions, i.e. monitoring the efficacy of a

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treatment by measuring changes in the presence of driver muta-tions before and after administration of a given drug or surgicalprocedure. This would greatly help doctors in assessing the effi-cacy of therapy on a patient-by-patient personalized basis.

In summary, the nanolock– and nanocross–nanopore sen-sors are emerging as a powerful technology with an ability todetect point mutations that occur at very low frequencies. Byintuitively observing the unzipping kinetics between nano-lock-bound mutated DNA molecules and wild-type moleculesin a nanopore or the unmistakable long-duration currentblocks associated with cross-links, we are able to differentiatethe mutated and wild-type gene sequences. Therefore, thissingle-molecule-based technology has a potential to be usedin cancer diagnostics, personalized medicine, and biomarkerdiscovery.

Funding

The manuscript was supported by the National Institutes of Health(ES021007 to KS Gates, GM114204 to LQ Gu, and HG009338 to KS Gatesand LQ Gu).

Declaration of interest

The authors have no relevant affiliations or financial involvement with anyorganization or entity with a financial interest in or financial conflict withthe subject matter or materials discussed in the manuscript. This includesemployment, consultancies, honoraria, stock ownership or options, experttestimony, grants or patents received or pending, or royalties. Peerreviewers on this manuscript have no relevant financial or other relation-ships to disclose.

ORCID

Li-Qun Gu http://orcid.org/0000-0002-8710-6160Kent S. Gates http://orcid.org/0000-0002-4218-7411

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