Digestion of Dynamic Substrate by Exonuclease Reveals High Single

Oct 31, 2018 - From the bulk fluorescence measurements, short perfectly matched (PM) substrate which is as a correct input can be effectively digested...
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Article Cite This: Anal. Chem. 2018, 90, 13655−13662

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Digestion of Dynamic Substrate by Exonuclease Reveals High Single-Mismatch Selectivity Yingjie Yu,†,‡,⊥ Liang Ma,§,⊥ Lidan Li,† Yingnan Deng,† Lida Xu,† Hua Liu,∥ Lehui Xiao,*,∥ and Xin Su*,†

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College of Life Science and Technology, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China ‡ Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States § Clinical laboratory, China-Japan Friendship Hospital, Beijing 100029, China ∥ State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China S Supporting Information *

ABSTRACT: The distinctive nuclease activity toward nucleic acid substrates enables various applications in analytical chemistry and dynamic DNA nanotechnology. λ Exonuclease is a widely used tool for the processing of PCR products, and DNA sequencing. This enzyme also shows promise for reducing the leakage (i.e., activation in absence of a correct input) in DNA-based analytical methods and nanotechnology due to its sensitivity to mismatches. However, the selectivity of λ exonuclease for single-mismatch in most applications is not high. Inspired by the increased specificity of dynamic probes in DNA nanotechnology, we enhanced the single-mismatch selectivity of λ exonuclease by using very short double-stranded DNA (dsDNA) as the substrate. From the bulk fluorescence measurements, short perfectly matched (PM) substrate which is as a correct input can be effectively digested, but the existence of single-mismatch drastically reduces the digestion rate. Real-time single-molecule kinetics analysis reveals that PM substrate can be selectively stabilized by the binding of λ exonuclease, which combines with the differential stability of transient hybridization of short substrates to yield high single-mismatch selectivity. An excellent selective assay for a single-nucleotide mutation in KRAS was demonstrated, which permits detecting this mutation from cell line at as low as 0.02%, holding potential for detecting rare mutations in circulating tumor DNA of early stage cancers.

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Much efforts have been made to prevent leaky hybridization reactions, such as DNA clamps,7 locked nucleic acids,8 discrimination cascade,9,10 and highly purified DNA strands.11 Genome modifying nucleases play a vital role in a variety of fundamental cellular processes, such as replication, repair, and recombination. With the development of DNA based sensing and DNA circuits, nucleasesparticularly site-specific nucleasesare extensively utilized for modifying DNA constructs in a precisely programmable way.12−14 Unlike site-specific nucleases, sequence-independent nucleases are typically avoided in manipulating DNA circuits in order to prevent nonspecific degradation.15 λ exonuclease is an Mg2+-dependent exonuclease that binds to the 5′-phosphorylated end of double stranded DNA (dsDNA) and processively digests the 5′phosphorylated strand releasing the complementary strand.16

he predictability of DNA base pair complementarity combined with a decrease in the cost of DNA synthesis have promoted the development of DNA based sensors and DNA computing.1 DNA based sensors employ the recognition capability of DNA to probe a variety of targets such as nucleic acids, proteins, small molecule metabolites, and cells.2 DNA circuits utilize a programmable network of reactions with nucleic acids to execute complex algorithms in order to process molecular information and computation.3 Nevertheless, a common problem in these systems is that hybridization reactions are “leaky”, it is difficult to completely prevent unwanted hybridization reactions from happening in the absence of a correct input.4,5 For instance, a leaky hybridization reaction may contain a single mismatch that only produces a small change in hybridization thermodynamics at room temperature, leading to false positive signals in many applications and wrong downstream manipulations in DNA circuits.6 Increasing single-nucleotide accuracy is therefore of great importance for DNA-based sensing and computing. © 2018 American Chemical Society

Received: August 30, 2018 Accepted: October 31, 2018 Published: October 31, 2018 13655

DOI: 10.1021/acs.analchem.8b03963 Anal. Chem. 2018, 90, 13655−13662

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Analytical Chemistry The ring-shaped homotrimer structure of λ exonuclease enables highly selective and processive digestion of 5′phosphorylated dsDNA substrates.16 These unique characteristics render λ exonuclease a promising tool for generating desired single stranded DNA (ssDNA) in a mixture of nuclei acid structures or species. It has been reported that the presence of mismatches inhibit the degradation activity of λ exonuclease to different extents, ranging from 4- to 320-fold.17 Ha and co-workers demonstrated that a single stranded bubble can trap the enzyme and hinder further digestion.18 λ exonuclease therefore holds promise to address the problem of leakage in nucleic acid reactions and circuits if the specificity can be made consistently high. Short nucleic acid duplexes (8−12 bp) hybridize only transiently at room temperature because of their low melting temperature.19−22 We therefore hypothesized that short dsDNA could be employed as a dynamic substrate to enhance the single-mismatch selectivity of λ exonuclease. We investigated the interaction of dynamic DNA substrates and λ exonuclease by combining bulk fluorescence measurements and single-molecule analysis. The bulk fluorescence measurements reveal that the dynamic substrate can be effectively digested by λ exonuclease and the existence of singlenucleotide mismatches drastically reduce the digestion rate. The single-nucleotide selectivity of the enzyme digestion reaction is superior to that of the hybridization equilibrium alone. The single-molecule kinetics analysis implies that the perfectly matched (PM) substrate can be selectively stabilized by the enzyme binding step, resulting in rapid digestion; the stability of the mismatched (MM) substrates is not increased by λ exonuclease binding. More enzyme binding events were observed in the presence of PM substrate rather than MM substrate. The high single-mismatch selectivity of digestion results from the combination of selective hybridization and enzyme binding preference. Taking advantages of the above observation, a highly selective method for the single-nucleotide change detection in KRAS gene was constructed allowing for detecting the mutant from cell lines as low as 0.02%. This study provides new insight into the interaction of substrate and enzyme and would find broad applications in DNA circuits, molecular diagnostics, and molecular motors.

quencher labeled DNA probes and 100 nM of their corresponding complementary strands were annealed in 1× λ exonuclease buffer containing 20 mM Tris-HCl, 100 mM (NH4)2SO4, 50 mM NaCl, 2 mM MgSO4, 0.1% Triton X-100 (pH 8.8). Twenty-six nM (40 U/mL) of λ Exonuclease was added. The fluorescence was recorded in the FAM channel of a real-time PCR cycler (Rotor-Gene Q, QIAGEN, Germany) at 37 or 25 °C with a time interval of 8 s. The degradation products were confirmed by denaturing gel. Objective Type Total Internal Reflection Fluorescence Microscopy (TIRFM) and Reaction Channel Set Up. Instrumentation was constructed for TIRFM using a Nikon inverted microscopy (ECLIPSE, Ti−U) equipped with a 100× magnification, 1.49 numerical aperture (NA) TIRFM objective (Nikon). For TIRF illumination, the laser of 520 nm was coupled into a single-mode fiber (Solamere Technologies). The fiber optic cable that delivers laser light to the microscopy was secured into a fiber launch fitted with an XY fiber holder mounted atop a micrometer-driven optical rail for Z adjustment (Thorlabs). Images were captured using electron multiplying (EMCCD) cameras (Andor). The pixel size of these cameras matches very well with the magnification offered by the 100× TIRF objective, giving a final resolution of 0.15 μm per pixel. Single-molecule imaging surfaces, quartz slide and cover slide were coated with 100% mPEG and a 10:1 mixture of mPEG and biotin-PEG, respectively. The reaction channel was constructed using two pieces of double-sided tape sandwiched between a quartz slide and glass coverslip as previously described.23 Protein Labeling. λ exonuclease with Mbp-ybbR tag was prepared according to the reported protocols.18,24 The purified λ exonuclease was labeled using Sfp synthase and CoA-547 dye conjugate (NEB) in 50 μL 1× SFP buffer (50 mM HEPES 10 mM MgCl2 pH 7.5) containing 1 μM Sfp Synthase, 10 μM CoA-547, and 3.28 μM λ exonuclease. The reaction mixture was gently agitated for 24 h at 4 °C in the dark. Excess dye was removed by extensive rinsing with washing buffer (25 mM Tris-HCl, 25 mM NaCl, pH 8.0,) using 500 μL concentrators. A labeling yield is ∼25% estimated by comparison of the absorption at 280 and 566 nm. Single-Molecule Characterization of DNA Transient Binding and the DNA-λ Exonuclease interaction. The channel was briefly incubated with 200 μL T50 buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) followed by 1 mg/mL streptavidin for 10 min, excess streptavidin was flushed out using T50 buffer. Next, 1 pM of 5′-biotinylated strand in the reaction buffer was injected into the channel and incubated for 10 min. The excess was flushed out by the reaction buffer for three times. Five nM of Cy3 labeled 5′-phosphorylated strand in oxygen scavenging system (2.5 mM PCA, 25 nM PCD, 1 mM Trolox)25 contained 1 × λ exonuclease buffer (using Ca2+ instead of Mg2+) was introduced into the channel. The fluorophore was excited by a 520 nm laser, and the fluorescence emission was detected with a filter of 593 nm by the EMCCD camera (200 ms, gain 20). For the singlemolecule enzyme binding assay, 26 nM of λ exonuclease was combined with the fluorophore-labeled DNA in the 1× λ exonuclease buffer (using Ca2+ instead of Mg2+), then injected into the channel. The single-molecule fluorescence was recorded immediately upon the enzyme injection. For the single-molecule digestion assay, Mg2+ contained buffer was used. All of the single-molecule assays were performed at 37 °C.

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EXPERIMENTAL SECTION The DNA, Enzyme, and Other Reagents. DNA strands used in this work were ordered from Sangon Co. (Shanghai, China) with HPLC purification. The sequences are listed in Supporting Information (SI) Table S1. λ exonuclease (5000 U/mL) and other proteins were purchased from New England Biolabs (Ipswich, MA). (3-Aminopropyl) triethoxysilane (APTES), 3,4-dihydroxybenzoate (PCA), protocatechuate dioxygenase (PCD), and Trolox were from Sigma-Aldrich (St. Louis, MO). mPEG-succinimidyl valerate (mPEG-SVA, MW, 5000) and biotin-PEG-succinimidyl valerate (biotinPEG-SVA, MW, 5000) were purchased from SeeBio Co. (Shanghai, China). Disulfosuccinimidyltartrate (sulfo-DST) was from Sangon (Shanghai, China). DNase/RNase free deionized water from Tiangen Biotech Co. was used for all the experiment. All of other chemicals are analytical grade without further purification unless mentioned. Monitoring the Degradation of Dynamic Substrates by Λ Exonuclease Using Fluorophore and Quencher Labeled DNA probe. Bulk fluorescence assays were carried out in 0.2 mL sealed PCR tube. 100 nM of fluorophore and 13656

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Figure 1. (A) The substrates digested by λ exonuclease monitored by fluorescence dequenching. F and Q represent the fluorophore (FAM) and quencher (BHQ1), respectively. (B) The fluorescence intensity response for the digestion of the 12-bp, fluorophore and quencher labeled substrate by λ exonuclease. (C) The fluorescence intensity response for the digestion of the stable substrate (21-bp) with λ exonuclease. In panel B and C, the solid and dash lines represent the reactions at 37 and 25 °C, respectively. The mismatch is C:A. The concentration of all substrates is 100 nM, and λ exonuclease is 26 nM. For the DNA sequence, see the denotation of SI Table S1.

Cell Culture, Genomic DNA Extraction, And Detection of the Mutant at Low Abundance. A549 and 1640 HEK-293T cell line were cultivated in RPMI 1640 and DMEM medium, respectively, supplemented with 1% Penn/Strep and 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air. Genomic DNA was extracted by using QIAamp DNA Micro Kit (QIAGEN, Germany) for downstream analysis. 240 nM of primers, 0.2 mM of dNTPs, 1.25 U/μL of Taq DNA polymerase and 0.002 ng/μL of the extracted DNA were mixed in the 1× λ exonuclease buffer for PCR amplification, which was carried out on GeneAmp 9700 thermal cycler (ABI, Foster City, CA) for 30 cycles with a procedure of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s. Next, lambda exonuclease (final concentration, 5 U/μL) was added to digest the 5′-phosphorylated strands which was extended by the 5′-phosphorylated primer for 20 min at 37 °C followed by inactivation of enzyme at 85 °C for 10 min. 100 nM of dual-labeled DNA probes and 100 nM of singlestranded PCR products were annealed in the above-mentioned reaction buffer to form dynamic substrates. Then, 26 nM of λ Exonuclease was added. The bulk fluorescence assay was carried out at 37 °C.

the length of dynamic substrate is set as 12-bp (predicted Tm 41 °C). The generally accepted zippering model guarantees the pairing of each base during the process of hybridization that is favorable for enzyme recognition.27 As shown in Figure 1B, the rapid fluorescence dequenching indicates that the dual-labeled strand in the dynamic substrate is degraded by λ exonuclease effectively. Recent study demonstrates that when a conjugated structure is covalently attached to a nucleotide of the stable substrate, the presence of a single mismatch at the 5′ side of the fluorophore modified base may accelerate the digestion of stable substrate by λ exonuclease.28 The mismatch is therefore set at the 3′ side of the fluorophore labeled nucleotide to suppress the digestion of the MM substrate. As expected, the existence of single-nucleotide mismatch in the middle of the dynamic substrate (4 bases from 5′-phosphorylated end) effectively hampers the substrate degradation (Figure 1B). As shown in Figure 1C, the single-nucleotide mismatch in the stable substrate (21-bp) only slightly reduces the degradation rate. Similar trends were also found when the assay was performed at 25 °C (dash line in Figure 1B and C). The singlenucleotide selectivity is higher at 37 °C by using the 12-nt probe in the typical reaction buffer (50 mM Na+ and 2 mM Mg2+, pH 8.8). It is interesting that the change of the degradation rate of PM dynamic substrate is not obvious at both 37 and 25 °C when reducing the number of base pairs from 21 to 12. The denaturing PAGE in SI Figure S1 confirms that the PM substrates can be digested effectively. The long MM substrate was partially digested and almost no digestion was found for the short MM substrate. As previously reported, the enzyme footprint of λ exonuclease is 13−14 nt which is longer than the dynamic substrate.16 Accordingly, our result suggests that substrates slightly shorter than the enzyme footprint can also be recognized and degraded by λ exonuclease or the footprint is overestimated in the previous report. Relation of the Hybridization Equilibrium and the Digestion Rate. The digestion by nucleases is a nonequilibrium process, whose rate is typically determined by equilibrium processes before catalysis.29 The hybridization equilibrium of short duplex is sensitive to internal and external factors. We therefore varied the number of complementary

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RESULTS AND DISCUSSION Digestion of Dynamic Substrate by λ Exonuclease Exhibits Single-Nucleotide Selectivity. λ exonuclease is an ATP-independent and Mg2+-dependent exonuclease that processively degrades dsDNA with a 5′-phosphorylated end into mononucleotides in the 5′ to 3′ direction.26 During the catalysis, λ exonuclease forms a symmetrical toroid homotrimer to accommodate dsDNA. Bulk fluorescence assay has been widely used to study the DNA nonequilibrium processes because DNA strand can be easily labeled by fluorophore or quencher with low cost. The principle of the bulk fluorescence assay is depicted in Figure 1A. A 5′-phosphorylated ssDNA labeled with fluorophore and quencher were annealed with its complementary strand to form dynamic substrates or stable substrates. As discussed elsewhere, the selectivity of hybridization is highest close to the melting temperature of the PM duplex.6 To ensure an appropriate hybridization equilibrium at the optimized working temperature of λ exonuclease (37 °C), 13657

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Analytical Chemistry bases, temperature, salinity, and the position of mismatch to construct the relation of the hybridization equilibrium and the digestion rate. As shown in SI Figure S2A-F, the reaction rates of all the PM and MM substrates increase with the number of complementary bases at both 37 and 25 °C. Salinity is expected to play a crucial role in the interaction of dynamic substrate and λ exonuclease because λ exonuclease is a Mg2+dependent exonuclease which exhibits high catalytic activity in the presence of Mg2+. Moreover, Mg2+ can enhance the stability of dsDNA especially for the dynamic substrates because divalent metal ion mitigates the electrostatic repulsion of the complementary DNA strands of a duplex.30 We found that the reaction rates for all types of dynamic substrate increase over the concentration of Mg2+ from 2 to 12 mM (SI Figure S2A-F) at both 37 and 25 °C. The optimized temperature of λ exonuclease is 37 °C. However, for the dynamic substrates, λ exonuclease exhibits higher activity at 25 °C in some conditions (e.g., 10-bp PM substrate with 2 mM Mg2+). Low temperature enhances the stability of the short duplex offsetting the reduce of enzyme activity. Most of the MM substrates are more sensitive to the salinity because their hybridization highly relies on high concentration of ions. The digestion rate at 25 °C is less sensitive to the high concentration of Mg2+ (e.g., from 6 to 12 mM) since high concentration of Mg2+ cannot bring additional stability. The position of mismatch in the duplex can also affect the duplex stability to a different extent. As previously reported, the equilibrium constant of a short dsDNA can vary from 102 to 105 nM due to the mismatch position.31 We introduced the C:A mismatch at different positions of the 12-bp PM substrate. Different reaction rates were found when the mismatch is set at different positions (SI Figure S3). The dynamic substrates with a mismatch in the middle (4 and 7 bases from the 5′phosphorylated end) exhibits lower reaction rate. The catalytic activity restores gradually by shifting the mismatch to the end. This result is consistent with the previous observation that the mismatch having a greater negative impact on duplex stability when it is closer to the middle of the duplex.31 In particular, the substrate with a mismatch 2 bases from the 5′phosphorylated end exhibits ∼10-fold digestion rate than the other mismatches. The mismatch close to the 5′-phosphorylated end might be helpful for the helicase of λ exonuclease which is necessary for initiating the digestion.32 On the other hand, the mismatch at the 5′ side of the fluorophore labeled base can accelerate the digestion because the label might interact with certain amino acid residues in the active site of λ exonuclease via π−π stacking interactions facilitating the digestion of the DNA strand.28 According to the above data, we constructed the relation of the hybridized fraction predicted by NUPACK33 and the digestion rate at 37 and 25 °C, respectively (Figure 2A and B). Positive correlations were found under these two temperatures. However, the correlation coefficients between the hybridized fraction and reaction rate are relatively low, which varies in a nonlinear relationship. These results suggest that the observable digestion rate cannot be attributed solely to the hybridization equilibrium, but is rather controlled by the synergistic function of DNA hybridization and the enzyme binding. Single-Molecule Characterization of the Interaction of Dynamic Substrate and λ Exonuclease. The information on the kinetics of hybridization and enzyme binding is useful to understand the selectivity of digestion. Bulk fluorescence assays do not allow us to directly measure the

Figure 2. Relation of the predicted hybridized fraction by NUPACK and the digestion rate at 37 (A) and 25 °C (B) (for data see SI Table S2 and S3). The parameters (DNA and ion concentrations) for prediction are the same as their corresponding digestion reactions (SI Figure S1 and S2).

kinetics of a reaction at equilibrium. Single-molecule kinetics analysis has transformed the study of biomolecule interactions since it records the key kinetic information and heterogeneity of biomolecule interaction.34 To elucidate the interaction of dynamic substrate and λ exonuclease, we first investigated the hybridization equilibrium of the dynamic substrates at singlemolecule level at 25 °C by total internal reflection fluorescence microscopy (TIRFM). As illustrated in Figure 3A, a 5′biotinylated ssDNA was immobilized on the PEGylated microscopy slide via biotin−streptavidin interaction. Upon the addition of the Cy3-labeled short ssDNA which transiently binds with the immobilized strand, the dynamic substrate reaches an equilibrium between single-stranded and doublestranded states. Due to the excitation geometry of TIRFM, the transient binding of dynamic substrates yields fluorescence-vstime trajectories of single molecules with high signal-to-noise (Figure 3B). The stochastic alteration of fluorescence signals between the ON- and OFF-states can be attributed to the binding and unbinding of the fluorescently labeled short probe from the immobilized strand.35 The single-molecule time trajectory of the hybridization is distinct from that the nonspecific absorption of Cy3-probe (SI Figure S4). Due to the PEG passivation of the surface, the fraction of nonspecific binding events is less than 10%. Fitting an exponential distribution to the dwell time histograms yields mean dwell time because the dwell time of a bimolecular under binding equilibrium are exponentially distributed.36 The PM substrates exhibit longer bound dwell time (SI Figure S5A and C) and shorter unbound dwell time than their corresponding MM substrates (SI Figure S5B and D). The photobleaching lifetime determined by the 21-bp stable substrate) is 78.4 s (SI Figure S6), which is much longer than the observed bound dwell times. 13658

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Figure 3. (A) Single-molecule kinetics analysis for the transient hybridization of dynamic substrate. The imaging surface was incubated with 1 pM biotinylated strands and the concentration of the diffusing strands is 5 nM. (B) Representative single-molecule time trajectories of the 12-bp PM and MM substrates. (C) Single-molecule characterization of the transient hybridization in the presence of the unlabeled λ exonuclease (26 nM). (D) Time trajectories of the PM (blue) and MM (black) substrates in the presence of unlabeled λ exonuclease. (E) The bound and unbound dwell time of the Cy3 labeled single strands with/without the unlabeled λ exonuclease. *** p < 0.001. Error bars denote s.e.m. from experiments performed in triplicate. All of the single-molecule assays were carried out at 37 °C in the presence of 2 mM Ca2+.

Figure 4. (A) Single-molecule kinetics analysis for the interaction of the dynamic substrates and the dye labeled λ exonuclease. The imaging surface was incubated with 1 pM biotinylated strands and the concentration of the diffusing strands is 5 nM. (B) Representative single-molecule time trajectories of the 12-bp PM and MM substrates in the presence of the CoA-547 labeled λ exonuclease (26 nM). CoA-547 is spectrally equivalent to Cy3. (C) The histogram of the measured fluorescence intensity of Cy3 from the dynamics substrates. All of the single-molecule assays were carried out at 37 °C in the presence of 2 mM Ca2+.

The unlabeled λ exonuclease was involved in the singlemolecule assay (Figure 3C). As shown in Figure 3D, the bound dwell time of the PM substrate is increased in the presence of λ exonuclease, however, for the MM substrate, bound dwell time does not show significant change in the presence of λ exonuclease (for exponential fit see SI Figure S8A−D). This implies that λ exonuclease can selectively bind and enhance the

We further investigated the influence of enzyme binding on the substrates. In order to suppress the digestion activity of λ exonuclease, Ca2+ was used in the buffer instead of Mg2+ because Ca2+ ion efficiently stabilizes the nucleic acid−enzyme interaction but does not support catalysis.29,32,37 We used the fluorophore and quencher labeled probe to confirm that the catalytic activity was greatly suppressed by Ca2+ (SI Figure S7). 13659

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Analytical Chemistry hybridization of those substrates with relatively higher stability (Figure 3E). To detect the enzyme binding in real-time, we further labeled λ exonuclease at the N terminus with CoA-547 fluorophore, which is spectrally equivalent to Cy3. The fluorescence change among three states suggests that the labeled λ exonuclease can bind the 10-bp PM dynamic substrate (Figure 4A). In contrast, the enzyme binding events are rarely found for the 12-bp MM substrate, and the enzyme bound time is much shorter than the PM substrates (Figure 4B). As shown in the intensity histogram, the probability of enzyme binding on the PM substrate is much higher than that of the MM substrate (Figure 4C). As reported previously, a stable substrate-λ exonuclease complex is the prerequisite for the processive digestion.28,32 The interaction of the PM substrates and λ exonuclease prolongs the lifetime of double strand, which allows the formation of the stable substrate− enzyme complex. However, the existence of mismatch hampers the effective binding of λ exonuclease and the subsequent formation of the complex. The presence of mismatch possibly distracts the 5′ end of the degradation strand from the catalytic center of the enzyme. The helicity of the duplex is important for processive degradation by λ exonuclease.32 The mismatch in a dynamic substrate amplifies the above effect rendering high single-mismatch selectivity of digestion detected by the bulk fluorescence. The digestion was also characterized at single-molecule level, from the microscopy images in SI Figures S9, the PM substrate can be digested more rapidly than the MM substrate. Highly Selective Detection of Single-Nucleotide Mutation. We compared the experimental single-nucleotide discrimination factors (DF) of all assay conditions in SI Figure S1, which is the ratio of the initial slopes of the PM and MM substrates digestion curves. The 12-nt probe exhibits highest DF in the presence of 2 mM Mg2+ at 37 °C (SI Figure S10A− C). To test the generality of single-nucleotide selectivity, we synthesized four types of 12-nt dual-labeled probes (for sequence see SI Table S1) to cover 12 types of singlenucleotide mismatches. All of the PM substrates exhibit much higher digestion rate than their corresponding MM substrates (SI Figure S11). We found that the DFs in our system are higher than the DFs that are derived from the ratio of the hybridized fraction of PM and MM substrates predicted by NUPACK (50 mM Na+ and 2 mM Mg2+) (Figure 5). Nucleases assisted strategies have been widely used for sensitive and selective DNA detection.38,39 For instance, the invader assay takes the advantages of the cleavage selectivity of Flap endonuclease to achieve allele discrimination.40 By combining the binary probe41 and RNase HII, Kolpashchikov and co-workers developed an enzyme-assisted assay for nucleic acids detection with real-time fluorescent signal readout rendering low limit of detection and discrimination ability toward a single-base substitution.42 Tumors are known to shed nucleic acids into the bloodstream, however, the mutation fraction in circulating tumor DNA (ctDNA) is very low, even lower than 0.1% during the early stage of cancer.43 In some nucleases aided strategies, the single-nucleotide selectivity is not high (the DFs are typically below 10), which is not enough for detecting low abundance mutant.44−46 This is mainly because the catalysis of nucleases is not sensitive to the mismatch in a stable substrate (Figure 1C). Due to the high DF, we speculated that our method can be employed for the detection of single-nucleotide mutation in low abundance. KRAS mutations widely exist in cancers.47 KRAS G12D

Figure 5. DFs for different types of mismatches. The experimental DF is the ratio of the initial reaction rate (calculation based on the first 100 s of the fluorescence curves) of the PM and MM substrates at 37 °C. The predicted DF is derived from predicted hybridized fraction ([dsDNA]/([dsDNA]+[ssDNA])) of PM and MM substrates by NUPACK (50 mM Na+, 2 mM Mg2+, 37 °C). The DNA concentration for prediction is 100 nM.

(c.34G > A) mutation in A549 cancer cell line was chosen as target for proving the feasibility toward biological samples. The wild-type DNA was from HEK-293 cells, which are predicted to only contain wild-type gene. Genomic DNA was extracted from the cells, PCR reactions were performed to quickly amplify the DNAs by using a 5′-phosphorylated reverse primer. The single stranded products were generated by λ exonuclease digesting the phosphorylated strand. The short probe was used to bind with the long products (Figure 6A). Because λ exonuclease can recognize the recessed 5′-termini,16 the hybridization of the long PCR product and the short probe can initiate the digestion. The probe design also allows the mismatch close to the middle of the dynamic substrates. The probes are fully complementary for the mutant and forms single mismatch with the wild-type. As shown in SI Figure S12, the 12-nt probe shows higher single-nucleotide selectivity than the 11-nt probe. By fixing the total concentration of target DNA, we adjusted the amount ratio of mutant to wild-type to prepare a serial of mixed samples with mutant at different abundances. As shown in Figure 6B and C, the mutant at an abundance as low as 0.02% can be effectively detected in the presence of wild-type. The initial rates of fluorescence restoration for these assays were measured, suggesting that the rate increases linearly over a range of the mutant ratio from 0.02% to 10% (Figure 6D). Owing to the featured interaction of dynamic substrate and λ exonuclease, the selective detection of mutant gene at low abundance is achieved. The use of interrelating dye or other cleavage detection methods such as gold nanoparticles44 is helpful to avoid the label of fluorophore and quencher reducing the cost of this method. Our study reveals that λ exonuclease can degrade short dynamic substrate with high single-nucleotide selectivity. The synergistic function of hybridization kinetics and λ exonuclease binding preference provides remarkable single mismatch discrimination capability. It was reported previously that λ exonuclease digestion comprises three phases: initiation, 13660

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Figure 6. (A) Schematic illustration of the generation of singlestranded PCR products and the binding of short probe and the products. (B) Fluorescence curves of the KRAS mutant at different ratios. (C) The curves of the low abundance mutant. (D) Linear relation of the initial fluorescence rate and the mutant ratio from 0.02% to 10%. The comparison of 0.02% and 0% mutant yields a pvalue of 0.003, confirming a significant difference. Lower fraction 0.01% only yielding a p-value of 0.08 is not shown. The assay was carried out at 37 °C, the total concentration of target DNA was fixed at 100 nM. 100 nM of the 12-nt dual labeled probe and 26 nM λ exonuclease were used. The mismatch is T:G. For the DNA sequence, see the denotation of SI Table S1. Error bars represent s.e.m. from triplicate experiments.

Figure 7. Proposed mechanism of the selective digestion of dynamic substrates by λ exonuclease.

broaden the applications of λ exonuclease in molecular biology and analytical chemistry.

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CONCLUSION In conclusion, the combination of dynamic substrates and λ exonuclease permits more accurate manipulation of DNA constructs and helps to address the problem of leakage in DNA-based sensing and logic circuit systems. Taking the advantages of the single-mismatch selectivity of this system, we developed a highly selective method for detecting a singlenucleotide KRAS mutation in DNA extracted from a cancer cell line at an abundance as low as 0.02%. Our method holds potential for detecting rare mutations in circulating cell-free DNA for clinical applications. This model holds potential to be extended to other nucleases. Dynamic DNA reactions as a smart therapeutics tool seeks broader applications in a living cell such as drug delivery, biomarker recognition, and subcellular structure imaging.1,49 Nucleases catalyze a variety of biological processes in cell. This study provides new insight into the interaction of enzyme and substrates, which might be helpful to transfer the success of dynamic DNA nanotechnology into the cell.

distributive, and processive.18 The initiation and distributive phase are nonproductive phases and their time scale is concentration-dependent, which can roughly cost 12 s when the concentration of λ exonuclease is 26 nM. During these two phases, frequent dissociation of λ exonuclease from the substrate was observed by Lee et al. Thus, during these first two nonprocessive phases, λ exonuclease is predicted to be sensitive to the stability of a transiently hybridizing substrate. As shown in Figure 4A, the bound dwell time of PM and MM substrates is comparable to the time scale of the two nonprocessive phases of digestion. Furthermore, the PM substrates are selectively stabilized by λ exonuclease binding, resulting in a longer bound dwell time, further increasing the probability of proceeding to the following digestion step. By contrast, the MM substrates are not stabilized by the enzyme, and typically dehybridize before the digestion step. Hence, we proposed that the combination of selective hybridization and selective stabilization of the substrate by λ exonuclease binding appears to give rise to the high single-mismatch selectivity of λ exonuclease for short substrates digestion (Figure 7). Some noncanonical properties of λ exonuclease were recently disclosed by Wu et al.28,48 5′-nonphosphorylated duplex substrates can also be digested by the enzyme as long as there is 2−3-nt overhang at the 5′ end. The digestion rate can be accelerated by modifying the 5′ end with hydrophobic group such as C6-spacer, which can be comparable with the 5′phosphorylated substrates. The labeled fluorophore at the 5′ nonphosphorylated overhang end can serve as a reporter and a good recognition group for λ exonuclease, permitting the facile design of fluorescence dequenching probe for analytical applications.48 These novel properties rely on the hydrophobic interaction of the amino acid residue at the catalytic center of the enzyme and the modified 5′ end of the substrates. The new findings of λ exonuclease combined with our work may

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03963. The sequences of oligonucleotides used in this work are listed in Table S1. The predicted hybridization yield of the substrates and the experimental digestion rates are shown in Table S2 and S3. The supporting figures are included (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(L.X.) E-mail: [email protected]. *(X.S.) E-mail: [email protected]. ORCID

Lehui Xiao: 0000-0003-0522-2342 Xin Su: 0000-0002-6629-9856 13661

DOI: 10.1021/acs.analchem.8b03963 Anal. Chem. 2018, 90, 13655−13662

Article

Analytical Chemistry Author Contributions

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These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank A. Johnson-Buck from University of Michigan for MATLAB analysis code. This work was supported by the National Natural Science Foundation of China (31600687, 21522502), Fundamental Research Funds for the Central Universities (1206009226, 12060092151), Beijing Young Scholar Funds (2016000020124G033), the 13th Five-Year major projects (2018ZX09721001).

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DOI: 10.1021/acs.analchem.8b03963 Anal. Chem. 2018, 90, 13655−13662