Probing DNA Hybridization Equilibrium by Cationic Conjugated

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Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation Zehao Li, Xu Zhou, Lidan Li, Shue Liu, Congshan Wang, Lina Li, Changyuan Yu, and Xin Su Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00870 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Analytical Chemistry

Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation Zehao Li,a,† Xu Zhou,a,† Lidan Li,a Shue Liu,b Congshan Wang,a Lina Li,a Changyuan Yua,* and Xin Sua,* a

College of Life Science and Technology, Beijing University of Chemical Technology, Beijing

100029, China. b

Department of Gastroenterology, China-Japan Friendship Hospital, Beijing 100029, China.

*

Corresponding author

†

These authors contributed equally.

Email: [email protected], [email protected] Tel: +86-10-64421335 Fax: +86-10-64416248

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Abstract Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging. However, the single-nucleotide selectivity is still challenging because the specificity of hybridization reaction is typically low at room temperature. We disclose an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation (SNM) by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer (CCP). Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance as low as 0.1%. Single-molecule fluorescence resonance energy transfer (smFRET) study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent, which can be an explanation for the high single-nucleotide selectivity. Due to the simple design of the probe and the stable brightness of CCP, highly selective mRNA in situ imaging was achieved in fixed cells. Melanoma cell line A375 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HegG2 that was not reported having the mutation at this point.

Keywords Cationic conjugated polymer; Single-nucleotide mutation; Short DNA probe; Fluorescence resonance energy transfer; Single molecule; mRNA imaging

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Introduction Single-nucleotide mutation (SNM) in genome has drawn great attention since it has proven associated with tumorigenesis1. SNMs in APC, KRAS, or BRAF genes have been classified as cancer-initiating events and thus represent promising biomarkers for molecular diagnosis of beginning cancer and its precursors2,3. Accurately identifying SNM can offer crucial information such as tumor status and therapeutic response. Sequencing techniques4 and selective PCR5,6 provides solutions for SNM detection. On the other hand, the predictability of DNA base pair complementarity combined with a decrease in the cost of DNA synthesis has facilitated the development of hybridization-based DNA sensor7 and DNA circuits8. Nevertheless, the specificity of stable hybridization reaction is limited because single-mismatch can only produce a tiny change in duplex stability at room temperature or physiological temperature. Much effort has been made to improve the selectivity of hybridization reaction. The concept of dynamic DNA nanotechnology has been extensively used to amplify the effect of single-mismatch in hybridization thermodynamics and kinetics permitting effective discrimination of mutant and wild type9-12. Several enzymes are able to manipulate nucleic acid structure in a highly precise way13,14, and the interaction of these enzymes and their substrates exhibits single-mismatch selectivity allowing sensitive and selective SNM detection15,16. The success of these methods enables detecting the mutation at low abundance in liquid tissue biopsy. However, the complicated design of these probes and the dependence on enzymes limit their applications, such as in situ imaging. Simple probe design and high signal-to-noise are the prerequisites for the SNM detection and imaging with high confidence. Very short DNA oligonucleotides (9-12-nt) can only transiently bind with their complementary strands at room temperature17,18. Single-molecule kinetics analysis demonstrated this hybridization kinetics is sensitive to single-mismatch19. Taking advantage of this property, DNA-point accumulation imaging in nanoscale topography (DNA-PAINT) have been developed for subcellular super-resolution imaging20 and biomarker counting21. However, it is not practicable to design a probe for SNM detection and in situ imaging because the hybridization yield is low resulting in poor signal-to-noise.

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Conjugated polymers (CPs) are synthetic macromolecules with large pi-conjugated backbones and delocalized electronic structure22. The remarkable light-harvesting and amplifying properties render CPs a promising tool for biosensing and bioanalysis23-25. Water-soluble cationic conjugated polymer (CCP) emerge as molecular photonic wire in a variety of colorimetric and fluorometric assays for proteins26, nucleic acids27, and even bacterial28. When CCP binds with a negatively charged target that is labeled with an energy acceptor, excitation energy efficiently migrates along the polymer backbone and transfers to the acceptor by fluorescence resonance energy transfer (FRET), resulting in the fluorescence amplification. Owing to the excellent ability of CCP, it holds potential for probing the hybridization equilibrium of very short DNA duplex. Here, we report a highly selective and simple method for detecting SNM and in situ mutant mRNA imaging by using CCP and short DNA probe. Taking advantage of the weak binding of short single-stranded DNA and CCP, CCP was used to distinguishing the hybridization equilibrium of perfectly matched (PM) and mismatched (MM) short duplex via FRET. Short DNA duplexes are sensitive to mismatch but their dissociation constants are typically high. Our system offers detectable FRET signal even the hybridization yield is low mainly because the CCP amplifies the fluorescence signal of the acceptor. Furthermore, single-molecule FRET (smFRET) reveals that the presence of CCP shifts the hybridization equilibrium of the short duplex to the hybridized state, which also contributes to the high single-nucleotide selectivity. CCP also prevents nucleic acids from nuclease digestion potentially enabling the applications in crude biological samples. This method was applied for in situ imaging of mRNA in different cell lines. The difference in the donor and acceptor channel reflects effective discrimination of mutant and wild type mRNA. Experimental Section Materials. All DNA strands used in this work were synthesized and purified by HPLC (Sangon Co., Shanghai, China) and their sequences are listed in Table S1. DNase I and Exo III were from from NEB (MA, USA). Chemicals for organic synthesis were obtained from Sigma-Aldrich (St. Louis, MO). mPEG-succinimidyl valerate (mPEG-SVA, MW, 5000) and biotin-PEG-succinimidyl valerate (biotin-PEG-SVA, MW, 5000) were purchased from SeeBio

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Co. (Shanghai, China). DNase/RNase free deionized water from Tiangen Biotech Co. (Beijing, China) was used in all experiments. Synthesis of the cationic conjugated polymer. As shown in Scheme S1, the cationic conjugated

polymer

poly[9,9’-bis(6’’-N,N,N-trimethylammonium)hexyl

fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)dibromide]

(PFBT)

was

synthesized

from

commercially available chemicals with four steps according to the previously reported method.29 The GPC curve was shown in Figure S1. In vitro DNA detection by the CCP. In a typical in vitro assay, to a 1.7 mL centrifugate tube, 20 µM of PFBT, 400 nM of target DNA, 400 nM of Cy5-labeled short DNA were incubated for 10 min at room temperature in a 10 mM phosphate buffer. Fluorescence was measured by the microplate reader. The fluorescence (Ex: 500 nm) was measured on a multilabel reader (EnVision, PerkinElmer, UK). TIRF-based smFRET experiments for characterizing DNA hybridization. TIRFM was constructed by using a Nikon inverted microscope (ECLIPSE, Ti-U) equipped with a 100×magnification, 1.45 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 microscope was secured into a fiber launch fitted with an XY fiber holder mounted atop a micrometer-driven optical rail for Z adjustment (Thorlabs). Single-molecule imaging surface was coated with a 10:1 mixture of mPEG and biotin-PEG. A single flow channel per slide was assembled using double-sided sticky tape and a glass coverslip. 1 mg/mL streptavidin in 1×T50 buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.5) was injected to the channel and incubated for 10 min at room temperature to capture DNA via the biotin/streptavidin interaction. Then 50 pM of biotinylated DNA and 3 nM of PFBT were added and incubated for 10 min. The excess of non-immobilized DNA and CCP were rinsed by flushing the channel with the T50 buffer. For the smFRET by using Cy3 as donor, 50 pM of the duplex of biotinylated single strand and the Cy3-labeled single strand was immobilized on the surface. 20 nM of Cy5-labeled short DNA probe in oxygen scavenging system (2.5 mM PCA, 25 nM PCD, 1 mM Trolox)30 phosphate buffer (200 mM NaCl, pH 7.4) was introduced into the channel. The fluorescence emission light was further separated into donor and acceptor signals 5



with a 630-nm dichroic mirror (Semrock) and was detected by the back-illuminated electron-multiplying charge-coupled device (EMCCD, Andor) with an integration time of 100 ms and with a total length of 200 s. Only the trajectories with single-step donor and/or acceptor photobleaching, or showing clear anti-correlated donor–acceptor dynamics, were selected for hidden Markov modeling (HMM) idealization. The dwell time of each state was exponentially fit. Fluorescence imaging of target mRNA in fixed cells. A375, A549, HEK-293T, and HepG2 cell line were cultured in 1640 medium supplemented with 1% Penn/Strep and 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2/95 % air. The cells were fixed by using 4 % paraformaldehyde for 20 min. The cells were incubated with 20 µM PFBT and 400 nM Cy5-labeled 9-nt probe in 1×PBS for 10 min followed by rinsing by 1×PBS. Fluorescence imaging was carried out using the Nikon inverted fluorescence microscope equipped with a mercury light source. The filter with 540 nm was used for excitation, and the filters with 590 nm and 690 nm were used for detecting the emission of PFBT and Cy5. The images were acquired using a 40×objective and recorded by the EMCCD (exposure time 50 ms, gain 20). Results and Discussion Hybridization-based DNA probe can bind with its target to form a duplex. The discrimination of SNM relies on the difference on the hybridization yield of PM and MM duplex. The single-mismatch only generates small change on hybridization thermodynamics in a long duplex but a large change in a short duplex (Figure 1A). The hybridization yield of long duplex is higher than that of short duplex. We plotted the relation of specificity and sensitivity that represents the hybridized fraction of PM duplex and the ratio of hybridized PM and MM duplex, respectively (Figure 1B, predicted by NUPACK31). Briefly, high sensitivity and high specificity cannot be achieved simultaneously. In the previous study, at the melting temperature of PM duplex, this is a good trade-off between specificity and yield9. The length of probe and salinity can be adjusted to achieve this at room temperature. In practice, the simplest way to employ hybridization reaction for detecting SNM is using binary probes32. A donor fluorophore-labeled single strand is stably hybridized with the targets, and an acceptor fluorophore-labeled strand 6


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serves as the signal probe. We used a 10-nt signal probe to gain compromised sensitivity and specificity because the melting temperature of the 9-bp duplex is close to room temperature according to the prediction. A commonly used fluorophore pair, Cy3 and Cy5, was labeled on the binary probes (Figure 1C). The fluorescence emission was recorded. However, neither the sensitivity nor the specificity is high as expected mainly due to the poor energy transfer capability of organic dyes and the electrostatic repulsion of the negatively charged DNA strands (Figure 1D). Water-soluble CCPs can bind with negatively charged nucleic acids in aqueous solution. The binding of fluorophore-labeled oligonucleotides with CCP allows for highly efficient energy transfer between the CCP and the fluorophore. In the previous study, CCP is believed to strongly bind with the negatively charged oligonucleotide and weakly bind with the dNTPs33. Probing the hybridization events by CCPs always relies on the use of peptide nucleic acid probes34. Taking the advantage of the fidelity of polymerase, the SNM detection is achieved by the FRET between CCPs and incorporated fluorophore-labeled nucleotide35. Inspired by the different binding affinity of mononucleotide and oligonucleotide, we speculated that CCP could selectively bind with the oligonucleotides with different length. To test this, we measured the FRET between PFBT and Cy5-labeled oligonucleotides with different length. The emission of PFBT and the excitation of Cy5 are overlapped (Figure S2). As expected, the FRET efficiency increases with the length of oligonucleotides (Figure 2A). It is exciting that the CCP exhibits weak binding affinity towards short oligonucleotides (9-11-nt). This property may permit CCP to probe the hybridization equilibrium of short duplex at room temperature. As depicted in Figure 2B, we also used the 9-nt Cy5 labeled probe, here the PFBT serves as the donor in FRET. The probe is fully complementary with the mutant sequence (BRAF 1799A) and forms single-mismatch with the wild type sequence (BRAF 1799T). The existence of single-mismatch largely shifts the hybridization equilibrium. The mutant and wild type yield greatly different FRET efficiency (Figure 2C). We used the blocker which is a 20-nt oligonucleotide and complementary with the PM target to verify this strategy. The inhibition of the transient binding of Cy5 labeled strand results in a great decrease in FRET efficiency (Figure 2C). The use of CCP provides amplifies the signal. The combination of CCP and short DNA probe permits high sensitivity and specificity for SNM detection. The low abundance of single-nucleotide mutation in some circumstance such as cell-free DNA requires the capability for distinguishing PM and MM in a mixed sample36. By 7



fixing the total concentration of target DNA, we adjusted the percent of mutant to prepare a serial of mixed samples. As shown in Figure 3A, the mutant at an abundance as low as 0.1% can be effectively detected in the presence of wild-type, and the ratio of acceptor/donor intensity increases linearly over a range of the mutant ratio from 0.1% to 2% (Figure 3B). The detection of low abundance mutation renders this method suitable for the application of liquid biopsies. As discussed above, the length of probe potentially affects the single-nucleotide selectivity. The use of longer probes (10- and 11-nt) reduces the discrimination capability (Figure S3). The longer probes may possess low specificity in hybridization reaction and may bind with CCP in the absence of target. Environmental robustness is crucial for a nucleic acid probe. Metal ion can enhance the stability of DNA duplex especially for the short duplex because they mitigate the electrostatic repulsion of the complementary DNA strands37. This typically renders the performance of hybridization-based probe sensitive to the environment. In our system, the presence of CCP potentially mitigates the electrostatic repulsion weakening the effect of metal ions. As expected, there is no significant change in FRET ratio by varying the concentration of Na and Mg (Figure S4). smFRET is a powerful tool to investigate the interaction of biomolecules38. The key dynamic information can be disclosed39. To further study the hybridization reaction in the presence of CCP, we characterized this system by smFRET on total internal reflection fluorescence microscopy (TIRFM). As illustrated in Figure 4A, the 5’-biotinylated target strand was immobilized on the PEGylated microscope slide via biotin-streptavidin interaction, and the PFBT was introduced to bind to the target through electrostatic attraction. Upon the addition of the Cy5-labeled short DNA probe which transiently binds with the immobilized target, the dynamic substrate reaches an equilibrium between single-stranded and double-stranded states. The binding and unbinding events yield a featured smFRET trajectory, the high FRET and low FRET states represent the double-stranded and single-stranded states, respectively (Figure 4B and C)40. The similar trajectories were also found by using the 10-nt and 11-nt probes (Figure S5). The dwell time of each state of a bimolecular binding equilibrium are exponentially distributed41,42. Fitting an exponential distribution to the dwell time histograms yields mean dwell time. The PM duplex exhibits longer binding dwell time and shorter unbinding dwell time than their corresponding MM substrates (Figure 4D and E). Increasing the length of dynamic 8


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substrate expands the binding dwell time and shortens the unbinding dwell time (Figure S6). This reduces the difference between the mutant and the wild type, which is consistent with the result observed via bulk fluorescence measurement. We also characterized the hybridization equilibrium of the binary probe system (Figure 1C) by smFRET. It is found that the dwell time of high FRET state is longer and low FRET state is shorter for the PM and MM duplex in the presence of CCP, and the difference is more significant for the PM duplex (Figure 5A and B). The CCP enhances the stability of short duplex. The presence of CCP shifts the hybridization equilibrium to the double strand state mainly since the electrostatic repulsion between DNA is mitigated. Another challenge for the application of the hybridization-based nucleic acid probe in crude biological samples is the non-specific degradation caused by nucleases43,44. The nucleases that digest DNA probe rapidly leading false-positive signals widely exist in plasma45 and cytoplasm46. Typically, the negatively charged backbone of nucleic acid substrate is recognized by nucleases, and the presence of metal ion facilitates the hydrolysis of phosphodiester at the active center of nucleases. Cationic polymer and surfactant were reported to protect nucleic acid from nuclease degradation, which makes these materials as efficient gene delivery tool47,48. We speculated that the CCP used in this work is able to protect the DNA probes. As shown in Figure S9A, there is no significant change in FRET of the 9-bp PM duplex in the presence of DNase, which is a non-specific endonuclease. Similarly, the nucleic acids were also protected from the digestion of Exo III (Figure S9B), which catalyzes the stepwise removal of mononucleotides from the 3′-hydroxyl end of duplex DNAs. The protection of nucleic acid probes by CCP is helpful to expand the assay in real biological samples. It is difficult to use conventional fluorescence in situ hybridization (FISH) technique for detecting a SNM on mRNA in cell because a long oligonucleotide probe will hybridize to the RNA despite the presence of a single-mismatch. Mismatch-sensitive strand displacement reaction was utilized to enhance the specificity of mRNA in situ imaging12. Li and co-workers employed the single-nucleotide selectivity of ligase for specific mRNA in situ imaging49. To test whether our method can be used for in situ probing mutation gene, we used human melanoma cell lines A375 with a well-known T to A mutation in the BRAF oncogene at position 1799 as mutant target50, and human liver cancer cell line HepG2 for wild type target. BRAF mutant was 9



rarely found in HepG2 cell line51. The PFBT and the Cy5-labeled 9-nt DNA that is fully complementary with the mutant mRNA were used. Strong fluorescence in Cy5 channel was observed in the assay for mutant target, suggesting high FRET and high hybridized fraction of mutant mRNA-DNA duplex (Figure 6A). Conversely, Cy5 channel is weak for the assay of wild-type target, suggesting low FRET and low hybridized fraction of wild-type mRNA-DNA duplex (Figure 6B). The capability of CCP for probing hybridization equilibrium of short duplex directly enables us to discriminate mRNA in situ with single-nucleotide accuracy. A 20-nt blocker strand was used in the imaging of mutant target. In this case, as expected the FRET efficiency is low because the blocker strand prevents the hybridization of Cy5 probe with target mRNA (Figure 6C). When the A375 cell was treated by RNase H (25 U/mL, 30 min) prior to imaging, no acceptor fluorescence was found (Figure 6D). To further verify the imaging specificity, the A375 cell was imaged by using a random Cy5-labeled DNA probe. As shown in Figure 6E, the weak fluorescence in the acceptor channel indicates there is no strong interaction between the polymer and probe. To test the generality, we imaged the mRNA of A549 cell line with KRAS G12D (c.34G>A) mutation which is widely exists in a variety of cancers52. HEK-293T cell was used as wild type target for imaging. As shown in Figure S10, this method also exhibits single-nucleotide selectivity for KRAS mutation. The success of selective imaging of different mutant mRNAs in fixed cell lines proves the generality of this method. Conclusion In summary, we successfully developed a method for SNM detection and imaging by using CCP to probe the hybridization equilibrium of short duplex. Owing to the specificity of short DNA and the signal amplification of the CCP, our method allows highly sensitive and selective SNM in vitro detection and in situ imaging. The SNM can be detected as low as 0.1% in a mixed sample. smFRET experiment suggests that CCP enhances the stability of the PM and MM short duplex to a different extent, which is an alternative reason for the high sensitivity. The CCP was also found having the ability to prevent the nucleic acid from degradation. This method represents a new approach for using hybridization-based DNA probe; it would find broad applications in DNA based sensing and intracellular imaging.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (31600687), Fundamental Research Funds for the Central Universities (12060090071, 12060046030), Beijing Young Scholar Funds (2016000020124G033), and the 13th Five-Year major projects (2018ZX09721001). Supporting Information The Supporting Information is available free of charge on the website. The sequences of oligonucleotides used in this work are listed in Table S1. The synthetic route of PFBT is shown in Scheme S1. The supporting figures are included.

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FIGURES AND CAPTIONS

Figure 1. (A) Comparison of the sensitivity and specificity of short and long hybridization-based probes. (B) The relation of sensitivity and specificity derived from probes with different length by the prediction of NUPACK. (C) The work principle of a binary probe system. A short signal probe is used for improving single-nucleotide selectivity. (D) Fluorescence emission of the binary probe assay. There is no significant acceptor signal in the mutant (PM) and wild type (MM) group. All DNA used in this assay are 400 nM.

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Figure 2. Probing the hybridization equilibrium of short duplex by CCP. (A) Fluorescence profiles of the PFBT and Cy5-labeled oligonucleotides and Cy5-labeled dNTP. (B) Scheme of the single-nucleotide discrimination by combining the CCP and the Cy5-labeled 9-nt DNA probe. (C) Fluorescence profiles of the CCP-Cy5 probe system in the presence of the BRAF V600E mutant strand (PM) or wild type strand (MM) or the blocker that is a 20-nt oligonucleotide and complementary with mutant sequence.

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Figure 3. (A) Fluorescence emission profiles in the presence of mutant at different abundances. (B) The ratio of acceptor/donor intensity increases with the percent of mutant sequence. Inset: linear relation of the ratio of the acceptor/donor intensity (I670 nm/I570 nm) and the mutant ratio from 0.1% to 10%. Error bars represent s.e.m. from triplicate experiments. The total concentration of target DNA was fixed at 400 nM, and the percent of mutant target varied.

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Figure 4. smFRET characterization of the DNA hybridization equilibrium in the presence of the CCP. Scheme of the smFRET assay (A). The complex of CCP-biotinylated target DNA was immobilized on the slide surface. The Cy5-labeled short DNA probe transiently binds with the target DNA. Representative donor and acceptor trajectories and FRET trajectories of the 9-bp PM (B) and MM (C) duplexes. Dwell time distributions of the 9-bp PM (D) and MM (E) duplexes. The solid lines represent a single-exponential fit, yielding an estimated mean dwell time.

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Figure 5. Dwell time of low FRET and low FRET states (green, CCP as FRET donor; orange, Cy3 as FRET donor), 9-bp PM (A), 9-bp MM (B). * p

Probing DNA Hybridization Equilibrium by Cationic Conjugated

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