Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for

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Nucleic Acid Self-Assembly Circuitry aided by Exonuclease III for Discrimination of Single Nucleotide Variants Zhuo Zhang, and I-Ming Hsing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03564 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

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Analytical Chemistry

Nucleic Acid Self-Assembly Circuitry aided by Exonuclease III for Discrimination of Single Nucleotide Variants Zhuo Zhang† and I-Ming Hsing †‡,* †Division of Biomedical Engineering ‡Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong *Phone :(852)23587131. Fax :(852)31064857. Email: [email protected] ABSTRACT: Robust and rapid discrimination of one base mutation in nucleic acid sequences is important in clinical applications. Here, we report a hybridization-based assay, exploiting nucleic acid self-assembly circuitry and enzyme exonuclease III (Exo III), for the differentiation of single nucleotide variants (SNVs). This one-step approach combines the merits of discrimination power of competitive DNA hybridization probes (probe + sink) with catalytic amplification assisted by Exo III. The phosphorothioate (PS) bonds modified on wide-type (WT) specific sink inhibit the Exo III digestion, thus subsequent catalytic amplification magnifies intended SNV targets only. The integrated assay exhibits improved SNV discrimination than hybridization probes relying solely on competition or amplification, and enables SNV detection at 1% abundance. Two frequent cancer-driver mutation sequences (EGFR-L861Q, NRAS-Q61K) were tested. Our strategy allows simple sequence design and can easily adapt to multianalyte SNV detections.

INTRODUCTION Single nucleotide polymorphism in the nucleic acid sequences can result in important functional consequences. SNVs in biological samples provide crucial information regarding disease status and therapeutic response, thus the development of a robust technology to detect trace SNVs has long been a focus of molecular diagnostics1-3. Watson-Crick hybridization rule is the underlying principle of nucleic acid testing. Much effort was made to achieve higher hybridization specificity, for example through the use of molecular beacons4,5 or triple-stem6 architecture DNA probes to reduce thermodynamic favorability of hybridization, or through the well-designed hybridization probes with the quantitative and accurate estimation of the associated thermodynamic and kinetic properties so as to obtain an optimal SNV discrimination7. Li and colleagues8 introduced competitive hybridizationbased approach using two reacting DNA probes (SNV-specific probe, WT-specific sink). The preferential hybridization reactions between the pairs of SNV and SNV-specific probe and WT and WT-specific sink improve reaction selectivity, resulting in high SNV discrimination. Recently, Chen and Seelig9 have reported a detection scheme utilizing competitive hybridization probes combined with entropy-driven catalytic amplification10. Their method demonstrated better SNV selectivity than the competitive hybridization process alone. The abovementioned assays rely on enzyme-free hybridization-based self-assembly among nucleic acid sequences. However, the needs for additional designed reactant oligo-strands to fuel the

cascaded circuits, e.g. strand-displacement-based target recycling, complicate the whole system and add burden for strand sequence design to avoid spurious interference10-12. Several enzymes for molecular biology research are widely employed as “nano-tools” to cut and paste nucleic acid substrates13. For example, exonuclease III (Exo III) as a versatile tool, can selectively digest the nucleotides from the blunt or recessed end of 3’-hydroxyl terminus of duplex DNAs, thus enabling the preferential cleavage of one strand and the spontaneous release of the other14. This salient feature of configuration-dependent Exo III activity has been exploited in several studies for signal amplification in DNA analysis15-19, metal ion detection20,21 and fueled stochastic DNA walker22,23. Most of the Exo III related work aimed to improve the signal sensitivity, but relatively fewer studies explored ExoIII to improve the specificity of the detection, which is a critical factor for differentiation of SNVs. In this work, we report a new hybridization-based assay leveraging the approach of competitive DNA hybridization probes mentioned above with the assistance of Exo III. The selective target-recycling was realized by the accelerated Exo III digestion on the SNV-specific probe upon each hybridization cycle, which has significantly amplified the signal of SNV over the mutated target (WT), enabling an improved SNV discriminating power than that of the DNA hybridization probes relying solely on competition or amplification. Notably, in this approach no extra nucleic acid reagents are needed and only minor modifications are required on the design of sink probe to inhibit the unintended Exo III digestion.

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(A). Competitive hybridization:

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(B). Exo III-assisted selective target-recycling:

(C). Fluorophore-contained X-Probe:

Figure 1. (A) Schematic representation of the competitive reaction pathway compositions; (B) integrated with Exo III-assisted selective SNV input recycling. Sink probe has phosphorothioate bonds (PS) modified on 3’ ends (indicated as * between bases) resistant to the cleavage of Exo III. For the toehold probe, other than the input binding domain, additional 2nt TT bases are appended on the 3’ end to protect the effective hybridization region from the non-specific digestion of Exo III (towards ssDNA) in the absence of inputs. For either SNV or WT inputs, they both possess 8nt overhang attached on 3’ ends to prohibit the Exo III nonspecific degradation. (C) Scheme of Exo III-assisted target recycling on X-probe. Sequences of the X-probe displayed here is designed for EGFR-L861Q (c.2582 T>A) mutation. ROX, carboxy-X-rhodamine; RQ, Lowa Black Red Quencher. X-probe has phosphorothioate bonds (PS) modification (indicated as * between bases) on 3’ ends of F, P and Q species that resistant to the cleavage of Exo III.

Figure 1 depicts the ExoIII-assisted reaction composition scheme of our approach, and EGFR-L861Q (c.2582 T>A) mutation sequence was used as an exemplary analyte for proof-of-concept demonstration. Assuming that a given sample either contains SNV or WT but not both, in the presence of standard competitive compositions containing SNV-specific probe and WT-specific sink, ideally the probe would preferentially bind to the intended target and produce signal, while the WT would hybridize with the sink. Both probe and sink sequences were designed based on toehold-exchange principle that enables reversible strand displacement, and high hybridization specificity across different mutation positions in the target sequence7. To magnify the signal ratio of SNV over WT, for each captured target upon hybridization, the SNV analyte, based on our design, would be preferentially released and recycled back for further competitive hybridization reaction, while the WT strand would not. We utilize the configurationdependent feature of Exo III that selectively cleaves mononucleotides from 3’ end of those preferred double-stranded substrates with blunt or recessed 3’ termini, while keeping the

ones with 3’-protruding ends (with extension of 4 bases or longer) resistant to cleavage11. Therefore, the SNV-specific probe was designed to possess 3’-protruding toehold that initially resists to Exo III degradation, and upon hybridization with SNV, it displaces the output signal strand, and forms a 2nt 3’-protruding duplex structure. The newly formed duplex becomes vulnerable to the Exo III digestion, which releases and recycles the target that can undergo multiple hybridizations with probes, dispatching more output signal strands and repeating the catalytic cycle. In contrast, WT recycling was suppressed by Exo III resistant 3’ end of sink modified by phosphorothioate (PS) bonds, where a sulfur atom was substituted for a nonbridging oxygen in the phosphate backbone of an oligonucleotide, rendering the internucleotide linkage resistant to Exo III degradation24. As a result, the PS bonds on 3’ end would inhibit the Exo III processibility on sink both before and after hybridization reactions, thus the WT inputs captured by sink would not be recycled thereby not amplifying signal. Statistically, the

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SNV input could have stochastic interaction with the sink and vice versa, the WT molecule also has possibilities bound to the probe due to the sequence similarity between the SNV and WT. Therefore, the reaction pathway can be catalyzed not only by the SNV but also the WT albeit with lower probability and slower kinetics, resulting in negligible recycled WT input. Considering the limited digestive activity of Exo III on ssDNA and 3’-protruding end of duplex DNAs, we rationally designed the reagents (8 nt overhang attached on 3’ end of SNV and WT, 2nt sacrificial TT bases appended on probe toehold region) to ultimately suppress the effects of Exo III non-specific digestion on the programmed hybridization reactions. To obtain quantitative characterization of the signal, conditionally fluorescent probes25 were used, in which the probe is initially quenched in fluorescence, and upon hybridization with an input strand, fluorescent signal could be detectable after the displacement of the quencher-linked strand. A previously reported X-probe architecture by Zhang et al. was adopted26,27. Its design consists of four strands (Fluorophore modified strand (F), Quencher attached strand (Q), C strand and P strand), and the sequence of the modified oligonucleotides (F and Q) can be decoupled from the target sequence. As a result, for different target analytes, the same F and Q species can be used and this largely reduces the synthesis cost especially for multianalyte detections. In principle, the hybridization reaction of the X-probe is similar to that of the toehold probe (Figure 1C), i.e. upon hybridization with target, the PQ subspecies is displaced and the quencher on Q is set apart from fluorophore on F, hence releasing fluorescence. The 3’ end of the newly formed complex (FC species hybridized with target, FC-SNV) is then available to Exo III digestion, in which the subsequent mononucleotide cleavage ends up releasing the target for catalytic reaction. The PS bonds were modified on 3’ ends of F, P and Q strands to protect the Xprobe architecture from unintended Exo III degradation. Two frequently observed cancer driver mutations: EGFR-L861Q (c.2582 T>A) and NRAS-Q61K (c.181 C>A) were tested.

1.2x excess of the top protector strand (i.e. O: C =1.2:1; Ps: Cs=1.2:1), in 1xTE solution supplemented with 12.5 mM Mg2+ as annealing buffer. To prepare the X-probe consisting of four strands (i.e. P, C, F, Q species), we mixed the strands with F: C: P: Q=1:1.2:3:5 ratio in 5xPBS buffer and annealed the mixture following the same protocol. All annealed probes and sinks were stored at 4°C until use. Native Polyacrylamide Gel Electrophoresis. To prepare 10% PAGE gel, 5mL 30% acrylamide/bis-acrylamide gel solution (29:1), 1.5 mL 10× TBE buffer, 105 µL 10% Ammonium persulfate (APS), 10 µL N,N,N’,N’tetramethylethylenediamine (TEMED) and 8.424 mL deionized water were mixed. The gel was polymerized for 45 mins at room temperature before soaked in 1x TBE buffer (pH=8.0) ready to use. 10 µL of each sample was mixed with 2 µL of 30% glycerol solution, and was loaded to the PAGE gel. The PAGE gel was running at a constant voltage of 100 V for about 100 mins at room temperature. After gel electrophoresis was complete, gels were stained in 2× Gel-RedTM Nucleic Acid Gel Stain solution (Biotium, USA) for 30 mins. Gel images were obtained by scanning gels in a Gel DocTM XR documentation system (Bio-Rad, USA). For fluorophorecontained PAGE results, the gels were scanned by a Typhoon TRIO System (GE Medical System, USA). Fluorescence measurement. A FS5 spectrofluorometer (Edinburgh Instruments Ltd. UK) and Aireka Cells quartz cuvettes were used to perform the real-time fluorescence measurements. X-probes were labeled with ROX fluorophore (excitation: 586nm, emission: 606nm). Slit bandwidth was set at 5.0 nm, and the fluorescence spectra was recorded immediately after reaction started, with an interval of 15s between each data collection. Reaction temperature was set to 37°C for all experiments and controlled by an external monitor. Prior to each measurement, the cuvette was washed 10 times in deionized water, 3 times in isopropanol, another 10 times in deionized water and finally dried with nitrogen.

RESULTS AND DISCUSSION

EXPERIMENTAL SECTION Materials and reagents. All oligonucleotides were purchased from Integrated DNA Technologies Inc. (USA). Functionalized strands (F and Q strands) were purified by HPLC and all other oligonucleotides were purified by standard desalting. The design and sequences of oligonucleotides used for probe/sink formation are given in the supporting information Table S1. 1xTE buffer was used to make stock solutions of all oligonucleotides with 100 µM concentration prior to use, and stored at 4°C. Serial dilutions were made based on this stock concentration with 1xTE buffer. The Exonuclease III and 10× NEBuffer 1 (1×NEBuffer 1: 10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1mM DTT, pH 7.0 at 25 °C) were purchased from New England Biolabs, Inc. and used without further purification. Probe preparation. Probe and sink molecules were prepared by using the same thermal annealing process, i.e. heating to 95°C for 10 mins and cooling to 20 °C over the course of 2 hours at 0.6% ramp down rate. Toehold probe and sink each consists of two strands, i.e. output signal strand O and C strand in toehold probe; Ps and Cs species in sink. The molecules were prepared by separately annealing the mixtures with

Investigation of Exo III-aided toehold probe in SNV detection. The assay was first experimentally tested for EGFRL861Q (c.2582 T>A) SNV detection. Figure 2 shows the native polyacrylamide gel electrophoresis (PAGE) result, in which the intensities of the probe, sink and output signal bands are directly comparable (30nt poly-T were appended on 3’ end of output signal strand for distinguishable gel band indication). We compared the bands mobility in the gel to explore the Exo III digestive effects on reactant strands in the absence of targets. The probe band slightly shifted to a higher-mobile position with the addition of Exo III compared to the one without (lanes 1, 2), while the mobility of sink band remained unchanged (lanes 3, 4). We believe this implies the Exo III nonspecific digestion towards on ssDNA and the slow-kinetic process can be largely suppressed by the Exo III-resistant PS bonds modified on the 3’ end of sink. The hybridization product can be revealed as the intensity of output signal band. For SNV detection, little hybridization product was observed utilizing only competitive probes (toehold probe + sink), while the signal band was largely intensified upon the Exo III addition (lanes 5, 6). Comparably, nearly no hybridization products were obtained from WT detection, both with and without

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Figure 2. Native PAGE (10% gel) result. The reaction mixture has final concentrations of 200nM target (SNV or WT), 200nM probe and 200nM Sink. 10 Units Exo III was added for catalytic target recycling. Reactions proceeded in 1xNEB buffer with 2.5mM Mg2+ at 37°C for 1 hour.

Exo III implementation (lanes 7, 8). The enhanced hybridization yield of SNV detection exploiting competitive probes with the aid of Exo III demonstrates the preferentially selective recycling of SNV over WT in our assay. Investigation of Exo III-aided X-probe in SNV detection. Figure 3 shows the PAGE result of X-probe adopted system targeting NRAS-Q61K (c.181 C>A) mutation. A clear red band representing the fluorescent FC-SNV complex was shown when SNV specifically hybridizing to the fluorophorecontained X-probe (lane 5). And upon the addition of Exo III, the newly formed FC-SNV complex was subject to digestion and smear fluorescent bands with lower molecular weights appeared as expected (lane 6). Surprisingly, another intensified fluorescent band with high molecular weights was also displayed in the upper side of gel (lane 6). We believe this is due to the shortened FC complex after the Exo III cleavage and spontaneous target release still having several bases paired with the PQ complex, forming a contemporary high-molecular weighted structure that moved slowly in the gel electrophoresis (explanation verified by other gel results, in the supporting information Figure S1). The summation of total observed fluorescent bands intensities in each lane indicates the relative hybridization yield of each reaction. And remarkably, the hybridization yield for SNV detection was amplified using Exo III together with competitive probes, while that for WT detection kept low even with Exo III addition (lanes 7, 8). This indicates the suppressed WT release from captured sink through modifying Exo III-resistant PS bonds on sink, suggesting the improved SNV discrimination of integrated assay (X-probe + sink + Exo III) than competitive probes as a result of the magnified hybridization yield difference of SNV against WT. Moreover, we obtained little fluorescent bands when X-probe alone reacted with Exo III (lane 2), which might be due to incomplete fluorescence quenching from the not well-annealed X-probe and the leakage fluorescent signal from the nonspecific digestion of Exo III. Quantitative performance comparison of three discrimination systems. Next, the time-based fluorescence response was characterized. The quantitative metric we utilized to compare the performance is the discrimination factor (DF): / , in which refers to the observed fluorescent signal when SNV hybridized to the X-probe and refers to the signal obtained from the reaction between WT and X-probe. Additionally, comes

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Figure 3. Native PAGE (10% gel) result of the integrated system targeting NRAS-Q61K (c.181 C>A) mutation. The reaction mixture has final concentrations of 200nM target (SNV or WT), 200nM X-probe and 200nM Sink. 10 Units Exo III was added for recycling intended target. Reactions proceeded in 1xNEB buffer with 2.5mM Mg2+ at 37°C for 2 hours.

from the background signal (incomplete quenched X-probe) plus the leakage signal induced by Exo III non-specific digestion on X-probe. For each analyte, we studied three different discrimination systems (Figure 4A), one based on the competition between SNV-specific X-probe and WT-specific PS bonds modified sink, a second consisting of Exo III-assisted signal amplification through target-recycling on X-probe, and finally an integrated system combining the two. Experimentally, for competitive probes alone system (X-probe + sink), upon the addition of either SNV or WT, it tends to show initial high fluorescent signal, and the signal gradually dropped to the equilibrium value over the course of 30 minutes (Figure 4(i)). This phenomenon was repetitively observed and we believe it is due to the competition between two reversible toehold exchange reactions. As the targets were the last to add in, immediately after pipetting, there is a high local concentration of targets that can quickly react with sink or X-probe before the solution is mixed. The WT targets reacted with X-probe need to slowly dissociate before hybridizing with sink that was initially not locally available. This process takes some time, in our case, 30 minutes to reach equilibrium. In comparison, for amplification alone scenario (X-probe + Exo III), the fluorescent signal obtained from either SNV or WT kept increasing, demonstrating that the reaction pathway can be catalyzed not only by SNV but also WT though with slower kinetics (Figure 4(ii)). For combined discrimination system (X-probe + sink + Exo III), we observed similar continuously increasing signal when targeting SNV as that in amplification-alone system, whereas the signal from WT was suppressed due to the presence of WTspecific PS bonds modified sink (Figure 4(iii)). Since the PS bonds modification would not affect the hybridization reaction of sink, the single base discriminating capabilities of three systems are directly comparable. The DF values were calculated accordingly (Figure 4(iv)), and we consistently achieved higher DF values (around 33~44) for combined discrimination system than competition or amplification alone scenario (around 5~6), given both EGFR-L861Q (c.2582 T>A) and NRAS-Q61K (c.181 C>A) mutations tested, which quantita-

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Analytical Chemistry (A). Reaction pathways:

(B). EGFR-L861Q (c.2582 T>A) i. Competition

ii. Amplification

iii. Competition with Amplification

iv. Discrimination factor

iii. Competition with Amplification

iv. Discrimination factor

(C). NRAS-Q61K (c.181 C>A) i. Competition

ii. Amplification

Figure 4. (A) Reaction pathways of three different discrimination systems; (i). A competitive composition consists of a SNV-specific Xprobe and a WT-specific PS bonds modified sink; (ii). An amplification system employs Exo III for target recycling; (iii). An integrated circuitry combines competitive compositions with Exo III-aided amplification; Experimental demonstration of three discrimination systems: (B). Time-based fluorescence response of three systems targeting EGFR-L861Q (c.2582 T>A) mutation. (C). Time-course fluorescence data of three systems targeting NRAS-Q61K (c.181 C>A) mutation. Green traces represent the signal obtained for SNV target, red traces for WT target and dashed black lines refer to the observed leak signal. Panel (iv.) shows the DF values calculated from the last measured 5 experimental data points and the error bars show the standard deviations across two repeats. All experiments were performed in 1X NEB with 2.5 mM Mg2+ at 37°C. The reaction mixture has final concentrations of 5nM target (SNV or WT), 10nM X-probe and 10nM Sink, with 10 Units Exo III addition for amplification circuitry and integrated discrimination system.

tively demonstrates the enhanced SNV discrimination by integrating competitive probes with Exo III-aided catalytic amplification. Selectivity test. To show that our methodology is useful for the detection of SNVs at low variant allele frequency (VAF) in a clinical setting environment, the detection selectivity of our assay was further assessed in a mixed sample. As seen in Figure 5, the combined system (X-probe + sink + Exo III) can easily differentiate SNVs at 1% VAF without adjusting conditions, demonstrating the high selectivity of our strategy by leveraging the specifically digestive feature of Exo III with competitive hybridization probes. Note that the X-probe and sink used here were not designed to achieve optimal thermodynamic parameters26; simulation-guided optimized probe

design combining with Exo III-assisted amplification would have enabled a lower VAF detection.

CONCLUSIONS We introduced and experimentally demonstrated a simple approach based on DNA hybridization probes, in which Exo III is employed in parallel with competitive probes (SNVspecific probe, WT-specific sink) to selectively amplify the signal in the presence of the intended target (SNV) over WT. We rationally designed the nucleic acid reagents and introduced phosphorothioate bonds (PS) modification on 3’ end of sink, to inhibit the unintended Exo III digestion. Both standard toehold probe and X-probe architectures were adopted and we

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REFERENCES

Figure 5. Fluorescence response of combined system to 500 nM WT (100% VAF, blue) and to 500 nM WT plus 5 nM SNV (1% VAF, green). Triplicate experimental traces are displayed. The reaction mixture has final concentrations of 7.5 nM X-probe and 750 nM Sink, with 10 Units Exo III addition. All experiments were performed in 1X NEB with 2.5 mM Mg2+ at 37°C.

chose two frequent cancer-driver mutations for proof-ofconcept demonstration. The developed Exo III-assisted SNV detection system shows better discrimination of single base difference in DNA sequences than hybridization probes relying on competition or amplification alone, and can easily enable SNV detection at low VAF (1%) in mixed sample. Additionally, the exploited Exo III-aided amplification strategy allows more flexible sequence design compared to entropy-driven amplifier. The Exo III-assisted target recycling relies on the specific cleavage of the involved nucleic acid substrates, instead of introducing extra fuel strands to drive the release of target, therefore relatively less strands are required, which avoids demanding efforts on reagent sequence design to exclude unfavorable mutual interactions, especially for multianalyte SNV detections. In summary, our work provides a simple way of combining nucleic acid tool enzymes with DNA hybridization probes, for rapid and reliable SNV discrimination, and may serve as a scalable strategy in multianalyte detections.

ASSOCIATED CONTENT Supporting Information Oligonucleotide sequences design (Text S1, Table S1); Explanation on the appearance of intensified high-molecular weighted bands after Exo III addition in Figure 3 (Text S2, Figure S1); Supplementary PAGE result of integrated system targeting EGFR-L861Q (c.2582 T>A) mutation (Text S3, Figure S2). The Supporting Information is available free of charge on the ACS Publications website.

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AUTHOR INFORMATION Corresponding Author *Phone :(852)23587131. Fax :(852)31064857. Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the Research Grants Council of the Hong Kong SAR Government for funding support (GRF# 16301817).

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Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for

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