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Adaption of Solid-state Nanopore to Homogeneous DNA Organization Verification and Label-free Molecular Analysis without Covalent Modification Zhu Zhentong, Ya Zhou, Xiaolong Xu, Ruiping Wu, Yongdong Jin, and Bingling Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03442 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Analytical Chemistry

Adaption of Solid-state Nanopore to Homogeneous DNA Organization Verification and Label-free Molecular Analysis without Covalent Modification Zhentong Zhu †, ‡,#, Ya Zhou †, §,#, Xiaolong Xu †,#, Ruiping Wu†, §, Yongdong Jin †,* and Bingling Li †,* †State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin, 130022, P.R. China. ‡University of Chinese Academy of Sciences, Beijing, 100049, China. §University of Science and Technology of China, Hefei, Anhui 230026, China * Email: [email protected], [email protected]. Phone: +86-431-85262008, +86-431-85262661 ABSTRACT: Recent advances have shown increasing designs of nucleic acid organizations via controlling thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nanotechnologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at single molecular level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of individual double helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultra-sensitivity of the nanopore technology, those concatemers difficult to observe on conventional electrophoresis image were brought to light. The translocation duration time also provided approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results were expected to promote deeper study and applications of nanopore technique in field of nucleic acid nanotechnology.

Recent advances in molecular engineering have brought to light several useful functions of nucleic acids other than genetic materials. For instance, recognition elements such as DNAzymes and aptamers enable recurring of diseases, detection and manipulation of biomolecules.1-4 DNA organization/assembly through origami technology has been proven useful for the purpose of precise positioning of molecules with subnanometer precision.5-7 Recently, a new class of DNA organization behaviours, also known as DNA catalytic circuits, has even exhibited great potential of enzyme-free amplification and bio-computing.8-13 While the design of nucleic acid organization has proceeded fast, the detection and characterization methodologies capable of probing these DNA nanostructures are still out of steps. The biggest challenge lies in the lack of nanoscale techniques within physiological environments.14 Being one of the best known label-free characterization approaches, atomic force microscopy (AFM) has been exclusively used for looking at the gross morphology of individual DNA structure.14-16 However, surface immobilization of DNA structures for AFM imaging may not reflect actual characteristics of the structures in the threedimensional solution. Moreover, its advantage has been overshadowed by the complicated sample handing process and subjective data selection. Although electrophoresis has become a quintessential apparatus in most laboratories, its accuracy and resolution are usually challenged by low sensitivity.

Strong electrical field or low salt may also potentially damage native structures or resulting in misreading. DNA organization also can be detected by label-related methods, such as spectroscopy and electrochemistry.10, 14, 17-21 However, in most of the circumstances, the expensive labeling or separation process can only suggest how many of two sequences have been assembled. Information about morphology usually remains ambiguous or completely unclear. Therefore, easier but effective methods for studying nucleic acid organization behaviours are still in urgent demand. In this research work, we explored the emerging nanopore sensing tactic to characterize in-situ DNA organization at single molecular level. The inspiration of this innovation came from previous discovery suggesting that when an inert polymer crosses through a submicron pore, the ion current of the pore may have a short decline since this polymer reduces the space of ion transport.22, 23 This kind of technique has exhibited unique advantages such as up to single molecule sensitivity, high-throughput capacity, high device portability, and sizeexclusive recognition. The last advantage suggests the duration time for polymer translocation can be sensitively associated with its length. Meanwhile the change of the current depends on the volume of the polymer occupying the inner space of the pore during the translocation process.24-37 This attribute is especially beneficial owed to the fact that most DNA organization processes are accompanied with significant changes in

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specie length, volume, or both that could be potentially discriminated from those short pieces of pre-assembly components. In our proof-of concept demonstration (Scheme 1a), we have successfully used the ion current of a conical glass nanopore (CGN) to distinguish the systems before and after onearm to four-arm hybridization chain reaction (HCR), a catalytic concatermer organization triggered by an oligonucleotide. 11 Due to the ultra-sensitivity of the method, those undetectable concatermers on traditional electrophoresis image were revealed. The duration time also provided approximate length diameter, and folding information for the concatermers. More importantly, the whole process happens in a completely homogeneous system without any covalently labeling or separating process. Besides promoting relevant researches for other size-enhanced organization systems, here CGN was proven to be a universal detector that could even detect the nucleic acid organization behaviors unable to generate enough products for reliable detection by other methods.

Scheme 1. (a) Scheme of a classic HCR pathway. Complementarity between numbered domains is denoted by an asterisk (*). (b) Illustration of dsDNA concatemers traversing through a CGN. (c) TEM image of CGN. The pore size calculation and reproducibility test was shown in Figure S1.

EXPERIMENTAL SECTION Materials and Fabrication of Nanopores are given in Supporting Information. One-arm HCR Reaction and Agarose Gel Electrophoresis. For all the HCR reactions, stock solutions of I0, H2, and H1 were diluted in 1×TNaK (20 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, pH 7.5) to four times their final concentrations (see legend). H1 and H2 were then respectively annealed at 95 °C for 5 min and cooled down to 25 °C at a rate of 0.1 °C/s before use. To start reaction, 10 µL H1, 10 µL H2, 10 µL 2M NaCl, and 10 µL different concentrations of I0 were mixed together, forming a standard 40 µL reaction liquid. After the liquid was incubated at 16°C for at least 6 h, 33 µL of reaction product was used for nanopore detection, while 5µL of it was loaded into agarose gel for electrophoresis detection. The 2% agarose gels contained 0.1 µL Gelred per ml of gel volume and were prepared by using 1×TAE buffer (40 mM Tris-Ac, 2 mM EDTA, pH 8.5). Agarose gels were run at 150 V for 30 min and visualized under UV light. Multi-arm HCR reaction was set up in a very similar way as that of one-arm reaction,

except that different hairpin substrates were used (see supporting information). Data Collection and Analysis. For DNA translocation, the nanopores were assembled into homemade horizontal type glass cells (Figure S2). The cell acted as trans reservoir and the inner cavity of glass capillary nanopore acted as cis reservoir. Two chlorinated silver electrodes were placed in each reservoir. Potential was applied to the electrode inside the nanopore. The ion currents were collected with current amplifier Axopatch 200B (Molecular Devices) using a low-pass Bassel filter of 10 kHz and digitized with DigiData 1440A digitizer (Molecular Devices) at a sample rate of 100 kHz. The RMS value of the measurement in this experiment is 1.1 pA. The DNA mixture (in 25 mM Tris-HCl, 640 mM NaCl, 5 mM KCl, and 0.5 mM EDTA, pH 7.5) was added into the trans reservoir. The current signal is processed using Clampfit 10.6 software (Molecular Devices). Real-time CHA Fluorescence Kinetic Reading. CH1, CH2 and F-Q mixture were, respectively, annealed at 95 °C for 5 min and cooled down to 25 °C at a rate of 0.1 °C/s before use. Kinetic reading of CHA was performed in 1× TNaK buffer. Final CHA components in a standard reaction mixture contained 100 nM CH1, 250 nM CH2, and 100 nM F-Q Reporter duplex with and without catalyst C1. In Reporter duplex, [F] was 100 nM, 1/2 that of [Q]. The fluorescence signal of 17 µL each CHA mixture was recorded every 1 to 3 min at 37°C on COYOTE Mini-8 Portable Real-time PCR system. Standard End-point CHA-HCR Nanopore Detection. Here the standard CHA reaction was carried out as above without addition of F-Q duplex. After 3 h incubation at 37°C, the reaction was added into contain amount of H1 (annealed), H2 (annealed), and NaCl, forming a total 40 µL reaction mixture including 100 nM CH1, 250 nM CH2, 1 µM H1, and 1 µM H2, with or without C1. The buffer condition was 1×TNaK with 500 mM NaCl. After 6 h incubation at 16°C, 33 µL of reaction product was used for nanopore detection.

RESULTS AND DISCUSSION Adaption of Glass Solid-state Nanopore to Verify HCR Concatemer in An Initiator-Dependent Way. Our initial model target was selected to be hybridization chain reaction (HCR), developed by Pierce group.11 The reason is that HCR has been engineered into various diversions and proven to be a very promising signal amplifier in many applications.13,17,1921,38 More importantly, HCR generates double stranded concatemers that can easily find references from traditional dsDNA markers.24-36 As shown in Scheme 1a, a traditional HCR reaction involves two 48-mer hairpin substrates (H1 and H2) being partially complimentary but not hybridized due to the heavy kinetic trap. Once a 24-mer initiator strand (I0) is introduced, it is expected to open one of H1 strands through toehold mediated strand displacement. Thereafter, this first step starts triggering a chain reaction wherein the two hairpins are alternatively unlocked to polymerize into a nicked duplex concatemer. For convenience, we abbreviated each concatemer as I0-(H1-H2)n. Previous AFM study validated that its volume should showcase consistency with any unfolded B-DNA helix, approximately 2 nm in diameter.25 Its length would be determined by the value of n. According to gel electrophoresis characterization, the concatemers under


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Analytical Chemistry each I0 concentration were actually not uniform, but ladderlike mixtures with n ranging from several to hundreds (Figure 1a).

Figure 1. Ability of nanopore for label-free and ultra-sensitive detection of HCR concatemers. (a) Agarose electrophoresis image before and after HCR reaction. From top to bottom: 10 Kb dsDNA ladder, 1 µM mixture of H1 and H2 with and without 0.1 µM I0, 2 µM H1 only. (b) 20 s continuous current vs time raw data extracted from 1~2 h electric recording of nanopore (at 200 mV) with the addition of HCR products derived from 2 µM H1 only, and 1 µM mixture of H1 and H2 without and with 0.1 µM I0. (c) Scatter plots of average nanopore current drop vs DNA translocation duration time. Note: Data populated in blue box including partially and completely folded events. (d) Typical current–time traces for three representative translocation patterns sampled from electrical recording.

Based on above parameters, we believed a conical glass solid-state nanopore (CGN, more accurately, nano-pipette) holding a few tens of nanometer diameters should be suitable enough to characterize HCR concatemers. Although the general signal-to-background ratios of solid-state nanopores are still far behind biological pores (e.g. aerolysin),30 they hold unique advantages of cheap and reproducible fabrication, easy storage, and size adjustability.39-43 Recently studies show that the electrochemically confined effect of nanopores provides the unique and rich information for the dynamic conformation of DNAs.44-46 Here following a few optimizations, we particularly chose the CGN with a reproducible diameter of about 10 nm ± 5 nm (Scheme 1b, 1c and Figure S1). Wider pore may lose resolution, while smaller one could be easily blocked during testing. We first prepared HCR systems comprising 1 µM H1 and H2, with or without I0. After 6 hour reaction, approximate 30 µL of reaction product was directly transferred into a nanopore set-up sitting at room temperature (Figure S2). As presented in Figure 1b, the pore was too inert to sense the translocation of either 48-mer H1 or H2 (H1 only and H1+H2), merely showing a relatively steady-state background ionic current during the entire recording window. While, once 0.1 µM I0 (0.1 × H1) was introduced, dense and sharp “needle-like” ionic current drops appeared upon back-ground current. These raw data threw light on the fact that the HCR organization generated products bulk enough to be recognized by the nanopore platform. When we zoomed into every individual current drop appearing in the current recording with 0.1 µM I0, three types of patterns appeared at highest frequency (Figure

1d), with profiling of square drop with amplitude of ~20 pA, square drop with amplitude of ~40 pA, and step drop with two amplitudes of ~20 pA and ~40 pA, respectively. Referring to previous study and our control experiments (Figure S3), they were precisely the three representative translocation states for a B-DNA double helix, which were, respectively, linear, folded, and partially-folded.47, 48 This was a more illustrative evidence to validate that the bulk products generated by HCR were double helixes. However, it should be noted that because only a small portion of organization products may translocate through and be counted by the nanopore, current drop signals may only reflect “existence” but not overall concentrations of concatemers. Thereafter, on the basis of a statistical scatter plot counted from more than 2400 current drops (in Figure 1c) together with respective duration time, we further derived a conclusion that the concatemers were mixtures covering a wide range of lengths (n values). Through the comparison the Gaussian fit received from a series of fix-length DNA markers (Figure S3f), large portion of concatemers dispersed within ~2 Kb (duration time less than 0.2 ms) to ~10 Kb (duration time close to 1 ms). In addition to obtaining above results consistent with electrophoresis (Figure 1a), we also observed a section of concatemers probably longer than 10 Kb (duration time≥1 ms), or even 20 Kb (duration time≥1.5 ms，Figure S3). It revealed the existence of trace amount of super-concatemers organized from more than 100 or even 200 H1 and H2, being consistent with previous AFM characterization20. They have previously been overlooked or ambiguously displayed by electrophoresis and other labeled readouts. Scatter Plots Enable Fingerprint-like Concentration Dependence of Initiator While turning to the concentration-dependence of I0, we apparently counted out considerable duplex-induced current drops under both 0.05 µM I0 and 0.01 µM I0 (Figure 2a) thathad shown merely ambiguous or non-observable bands on gel image. The scatter plots (Figure 2b-2d) indicated the length distribution of concatemers under 0.05 µM I0 was quite similar as that under 0.1 µM I0. It was conceivable because the two I0 concentrations were so close. While under 0.01 µM I0, on average, much shorter (< 20 Kb) concatemers were detected. In theory, the average n of concatemers should be in inverse proportion to the molar concentration ratio between I0 and H1 ([I0]:[H1]). Here the opposite result achieved could be explained by a reported “inhibitor ultrasensitivity” mechanism (Figure S4).18 That said when the initiator was much less than the substrates (H1 and H2), the small fraction of imperfectly formed H1 and/or H2 may lead to early termination of the HCR reaction once they are preferentially incorporated into the growing HCR chain. Above experiments validated the ability of nanopore in delivering ultra-sensitive detection. Therefore, it could be employed for the purpose of testing the HCR organization in the presence of much less substrate (H1 and H2) and initiator (I0). For instance, when H1 (and H2) concentration (e.g. 10 nM) was reduced to be non-observable on electrophoresis, nanopore results could still verify the effective formation of concatemers in presence of as low as 2 nM I0 (Figure S5).



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single set of CHA has been proven to be a universal transducer capable of recognizing both nucleic acid (ssDNA and RNA) and non-nucleic acid targets (e.g. small molecules and proteins),10, 13, 18, 19 the nanopore sensing platform could, thereafter, potentially detect these targets as well, without changing either CHA or HCR sequences.

Figure 2. Sensitive concentration discrimination of nanopore in the way like “fingerprinting”. (a) Agarose electrophoresis image of HCR products under different concentrations of I0. (b-d) Scatter plots and related all-point current drop histograms under 0.1 µM I0 (b), 0.05 µM I0 (c), and 0.01 µM I0 (d) at 200 mV. The populations of “Linear” and “Folded” in the scatter plots were shown by the color boxes. Note: Data populated in blue box including partially and completely folded events. In this experiment (b), (c), (d) were performed with the same pore.

As demonstrated above, the electronic raw data of nanopore are able to provide a sequence-specific “Yes-or-No” answer for the existence of origination initiator, I0. What’s more, patterns of scatter plots could distinguish different I0 concentrations in the same way as that of a “fingerprint”, no longer merely relying on different magnitudes of signals. Using HCR Concatemer as A Universal Non-covalent Tag in Detection of Small Inputs Making use of above demonstration and through employing HCR as an amplifier and bridge, the nanopore sensing platform could be further turned to a label-free and separation-free analytical tool (without any covalent modification) in order to sense small oligonucleotide inputs (such as I0) or even other organization behaviours that don’t generate nanoporedetectable huge products. In an example, this concept was realized with the use of linking HCR with another upstream enzyme-free organization amplifier, catalytic hairpin assembly (CHA). CHA also involves two hairpin substrates (named CH1 and CH2) and two successive toehold-mediated stand displacement reactions induced by a single stranded oligonucleotide (named C1, Figure 3a).9,10 While being different from HCR that produces elongating concatemers, CHA just enrich es CH1-CH2 duplexes. To enable the nanopore platform to recognize these “tiny” 53 bp CH1-CH2 duplexes, we thoughtfully incorporated above HCR initiator (I0, illustrated as domain 1-2) into CH1. It could be activated to trigger HCR only following the formation of CH1-CH2 duplex. From end-point recording (following the whole CHA-HCR coupling, Figure 3b), reaction with 5 nM C1 generated much denser and larger current drops as compared with that without C1. It indicated that CHA indeed generated effective HCR initiators in a C1dependent mode. After our deliberate design (See Figure S6), the CHA reaction in response to C1 could, in theory, generate 0.05 - 0.1 µM CH1-CH2 (HCR I0), while reaction in the absence of C1 could only generate less than 0.01 µM CH1-CH2 (HCR I0) in the same time course. Therefore, the reactions with and without C1 could be more reliably distinguished through the very different “fingerprints” (Figure 3c). Because

Figure 3. Ability of nanopore to universally sense small-scale organization behaviours or small targets. (a) Scheme of CHA cascading with HCR system. (b) 20 s continuous current vs time raw data extracted from 1~2 h electric recording of nanopore (at 200 mV) in presence of CHA-HCR products derived from reactions without and with 5 nM C1. (c) Scatter plots for reactions without and with 5 nM C1.

Adaption of Glass Nanopore to Verify Spatial DNA Organization with More Sophisticated Molecular Design. Following the proving that nanopore characterization is sensitive, label-free, and target friendly, the sensing platform was further applied for homogeneous verification of spatial DNA organization with more sophisticated molecular design. As shown in Scheme 1a (inset) and Figure 4a, the lowest theoretical diameter of the HCR concatemer could be gradually increased when the organization was varied from traditional one-dimension (one-arm) to multiple-dimension (two-arm, three-arm, and four-arm).The design could be realized via hybridizing one to four HCR initiators onto one ssDNA linker (Figure 4a).17 However, in either dye-staining (Figure 4b) or fluorescence label14 based electrophoresis detection, the four kinds of HCR reactions didn’t display much difference other than the speed to consume initiator. The volume or diameter information regarding each group of organization concatemers was totally missing. When nanopore was used instead, the average current-drop induced by concatemers produced from one-arm HCR to four-arm HCR was gradually increased, at big amplitude (Figure 4c-4f and Figure S7b-S7e). To the best of our knowledge, this is the first image (e.g. AFM)-free evidence for the diameter differences among the four groups of HCR concatemers. It further demonstrated the promising potential of nanopore technique in nucleic acid organization behaviour study. In detail, one-arm concatemers (Structure 1) resulted into an intensive current drop value at around 200 pA that showed consistency the fixed length DNA marker(Figure S8). The current drop of two-arm (Structure 2) and three-arm concatemers (Structure 3) brought forth an intermediate value from 200 pA to 400 pA. The four-arm concatemers (Structure 4) outputted the most of current drop value at around 1000 pA.


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Figure 4. Ability of nanopore to discriminate organization behaviours with more complicated folding. (a) Scheme of multiple-arm HCR pathway. (b) Agarose electrophoresis image of multiple-arm HCR products. (c-f) Structure, raw current traces, scatter plots and contour plots produced by one-way HCR (c), two-way HCR (d), three-way HCR (e), and four-way HCR (f) at 1000 mV. Note: (c), (d), (e), (f) were performed with the same pore. The event frequency in (c), (d), (e), and (f) is, in respective, 63 s-1, 72 s-1 , 84 s-1; and 82 s-1.

The non-linear increased current drop value may account for electrostatic repulsion between each assembly chain of concatemer. It should also be taken into consideration that here to push the huge multi-arm concatemers into the pore, 1000 mV voltage bias was chosen instead of the 200 mV put to use in previous experiments. Both current baseline and current drop values under different voltage bias are incomparable.49, 50 CONCLUSION In this research work, we brought forth the first proof-ofconcept demonstration with the use of nanopore sensing platform as a label-free tool for the characterization of DNA organization together with its derivative targets. Taking HCR and CHA as examples, it was found functioning like but more convenient in comparison with AFM, the nanopore sensing platform may detect the formation of individual concatemer via counting and profiling its translocation event. Functioning like but more sensitive as compared with gel electrophoresis, the nanopore sensing platform can deliver rough length and structure information through two parameters of current drop amplitude and duration time. Because of the ultra-sensitivity

of the method, the nanopore sensing plat-form may also reveal organization events having been blinded by electrophoresis as well as other detection methods. In addition, the whole characterization can proceed in homogeneous solution containing the same condition with organization process, which at the highest degree avoids the damage possibly induced by immobilization (e.g. electrochemistry detection), probe labeling (e.g. fluorescence detection), infinite dilution (e.g. AFM detection), and strong electricity field (e.g. electrophoresis). Despite being a preliminary exploitation, above results and conclusion were supposed to be suitable for many other nucleic acid organization systems. Extensive researches and applications on relevant fields are thus anticipated. In the meantime, deeper studies on many important factors like pore source, pore size, and transmembrane potential are also encouraged as they are more likely to play great roles in adjusting detection resolution and dynamic ranges.

ASSOCIATED CONTENT Supporting Information



Materials, methods, sequences. Figures for pore-size analysis, pore set-up, pore characterization on dsDNA markers, CHA verification, and gel images of different HCR systems. This material is available free of charge via the Internet at http://pubs.acs.org.

(20) Wang, F.; Elbaz, J.; Orbach, R.; Magen, N.; Willner, I. J. Am. Chem. Soc. 2011, 133, 17149-17151. (21) Wang, Q.; Pan, M.; Wei, J.; Liu, X.; Wang, F. ACS Sens. 2017, 2, 932-939

AUTHOR INFORMATION

(22) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770-13773.

Corresponding Author

(23) Porath, D.; Bezryadin, A.; Vries, S. de; Dekker, C. Nature 2000, 403, 635-638.

*[email protected]; *[email protected]

Present Addresses

(24) Dekker, C. Nat. Nanotech. 2007, 2, 209-215. (25) Howorka, S.; Siwy, Z. Chem. Soc. Rev. 2009, 38, 2360-2384.

†State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin, 130022, P.R. China.

(26) Venkatesan, B. M.; Bashir, R. Nat. Nanotech. 2011, 6, 615-624. (27) Reiner, J. E.; Balijepalli, A.; Robertson, J. W. F.; Campbell, J.; Suehle, J.; Kasianowicz, J. J. Chem. Rev. 2012, 112, 6431-6451.

Author Contributions

(28) Hou, X.; Guo, W.; Jiang, L. Chem. Soc. Rev. 2011, 40, 23852401.

#These authors contributed equally. The authors declare no competing financial interests.

(29) Ying, Y.-L.; Zhang, J.; Gao, R.; Long, Y.-T. Angew. Chem. Int. Ed. 2013, 52, 13154-13161.

ACKNOWLEDGMENT

(30) Cao, C.; Ying, Y.-L.; Hu, Z.-L.; Liao, D.-F.; Tian, H.; Long, Y.T. Nat. Nanotech. 2016, 11, 713-718.

Notes

We thank groups of Hongda Wang (Changchun Institute of Applied Chemistry), Hang Xing (Hunan University) and Hao Pei (East China Normal University) for critical help in HCR characterization. This work was supported by National Natural Science Foundation of China (No. 21505129, 21675146, 21175125) and Natural Science Foundation of Jilin Province 20160101296JC.

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