Skip to main content
NCBI home page
ACS Central Science logoLink to ACS Central Science
. 2020 Jan 9;6(1):76–82. doi: 10.1021/acscentsci.9b01129

Direct Quantification of Damaged Nucleotides in Oligonucleotides Using an Aerolysin Single Molecule Interface

Jiajun Wang †,‡, Meng-Yin Li †, Jie Yang ‡, Ya-Qian Wang ‡, Xue-Yuan Wu †,‡, Jin Huang §, Yi-Lun Ying †, Yi-Tao Long †,*
PMCID: PMC6978832  PMID: 31989027

Abstract

graphic file with name oc9b01129_0006.jpg

DNA lesions such as metholcytosine(mC), 8-OXO-guanine (OG), inosine (I), etc. could cause genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remains a challenge due to massive varieties and less distinguishable readouts for structural variations at the molecular level. Moreover, standard amplification and labeling hardly work in DNA lesion detection. Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor lesion into distinguishable electrochemical readouts. Compared with previous single molecule sensing interfaces, the temporal resolution of the K238Q aerolysin nanopore is enhanced by two orders, which has the best sensing performance in all reported aerolysin nanopores. In this work, the novel K238Q could discriminate directly at least three types of lesions (mC, OG, I) without labeling and quantify modification sites under the mixed heterocomposition conditions of the oligonucleotide. Such a nanopore electrochemistry approach could be further applied to diagnose genetic diseases at high sensitivity.

Short abstract

Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor DnA lesion into distinguishable electrochemical readouts.

Epigenetic modified DNA nucleotide causes protein dysfunction inducing tumors or even cancer diseases. Commonly, mass spectrometry or fluorometry are the approaches to identify the modified nucleotide bases. In a typical HPLC/mass spectrometry analysis,1 lesion segments could be discriminated at high resolution; however it requires tedious HPLC purification of the specific lesion part. Other reagents or labeling processes based on fluorometry provide feasibility as well as accuracy, but lack generalization to each lesion variant. In addition, the lesion types, with the most common instance being methylated cytosine (mC), occurred at a frequency of one per 1 million repeats, which indicates ultrahigh sensitivity is required.2 Because of the massive types of lesions and the low occurrence, the sequencing and identification of the lesion unit even in a heterogeneous condition are too challenging for conventional approaches.

A generalized, label-free single molecule technique is required to address these limitations, and the nanopore electrochemistry approach is presented here. The nanopore can be the most prominent tool for DNA sequencing and lesion nucleotide detecting. In a proof-of-concept experiment,103 a single-stranded DNA molecule is driven through α-hemolysin under an electric field. Transient ionic fluctuation is obtained to directly measure the polynucleotide length. More recently, the nanopore catalyzed by phi29 DNA polymerase is engineered allowing real-time detection of numerous nucleotide additions.3 The nanopore is also engineered by site-directed mutagenesis to slow down the DNA translocation owing to stronger noncovalent interaction allowing base identification.4 Nonetheless, none of these techniques achieved lesion detection in a mixed condition, owing to the few contributions from the single lesion occurrence in terms of mass, charge, or structure.

Currently, more powerful nanopores are presented, for instance, MspA,5 ClyA,6 CsgG,7 aerolysin,8 YaxAB complex,9 FraC,10 OmpG,11 Lysenin,12 CymA,13 FhuA,14 etc. Alternatively, careful design of nucleotide chains forms a hairpin structure or primer that could hook the segment inside the nanopore lumen. This approach allows the oxidative derivatives and even mispairing domain to fall into the sensitive region to be detected.15,16 Among the above-mentioned nanopores, aerolysin from Aeromonas hydrophila as one of the smallest pore owes ∼1.1 nm pore diameter.17 Prominently, wild-type (WT) aerolysin could identify a single base difference8 and single methylated cytosine18 at the end of a homonucleotide mixture solution. Nevertheless, the detection of multiple lesion types in a heteronucleotide mixture is still challenging.

It has been reported that both interaction and steric effects between the analyte and sensing interface in the nanopore determine the sensitivity.19 The space confined by the nanopore should enhance the weak interaction including the H-bond, van der Waal force, and electrostatic force so as to improve the single molecule probing capability.20 By testing with a model nucleotide segment (dA4), the residence time with prominent mutants such as K238E and K238G is 20 and 50 times longer than the WT, generating higher sensitivity. However, according to the statistical analysis of residence time and I/I0 for lesion nucleotides, the overlapped scatter distribution suggested the inefficient separation ability with these mutants (separation ability, denoted as S, correlates with the half-peak width following the previous protocol28). Therefore, we challenged the aerolysin nanopore to discriminate mixtures of more types of lesion units at different positions of a heteronucleotide. So far, a series of aerolysin nanopores, e.g., K238E,21 K238F,22 K238G,22 R282G,23 Q212R,23 N226Q,23 K238C,24 K238Y,24 K238C,25 and other aerolysin nanopores26,27 are designed, which attempted to discriminate a single nucleotide in a heteronucleotide, at a single molecule level. Nonetheless, no such mutant provides sufficient sensing ability to discriminate multiple lesions in a heteronucleotide. Herein, we present the best mutant aerolysin, K238Q, to quantify lesion nucleotides, which shows the strongest sensing capability among all the reported biological nanopores in nucleotide detection. To consider the weak interaction between each base and “key” amino acid (i.e., K238), glutamine (Q) was then chosen due to its strong interaction with nucleotide bases.29 Under constant voltage (+100 mV), the K238Q constantly opens at 52 ± 2 pA (for experimental and characterization detail, please refer to Supporting Information, Figures S1–S2; I–V relation refers to Figure S3). When applying the model nucleotide segment, dA4, K238Q reads the residue current and residence time as 34.5 ± 0.5 pA (I/I0 = 0.58) and 122 ± 19 ms respectively at 120 mV, which induces a half-peak width 0.5 pA. Comparing WT and all the mutants, the time domain resolution reported so far has reached the best for aerolysin, leading to an enhanced temporal resolution for single nucleotide discrimination by two orders. In this content, we demonstrate that the K238Q nanopore could discriminate three types (mC, OG, I) and quantify modifications under a mixed heterocomposition condition.

Figure 1.

Figure 1

Open in a new tab

Aerolysin nanopore measuring modified oligonucleotide. The aerolysin is composed of seven monomers, and the K238Q contains seven mutant sites (7K238Q). The modified nucleotides 8-oxo-guanine (blue), inosine (black), and 5-methylcytosine (red) at different positions on the model nucleotide segments are shown as a scheme. (A) A typical K238Q aerolysin inserted into an artificial lipid bilayer with substrates added from the cis side. The implementation of (B) additional zoomed-in diagram (red box in (A)) showing the position of the 238 site inside the aerolysin lumen and the sensing mechanism to dA4. The electrochemical signals from dA4 with WT (left) and K238Q (right) aerolysin are given. (C) The scatter plot shows the event distribution of dX4 (X = A, C, T) with WT (left) and K238Q (right). Residence time (τ) is prolonged two orders of magnitude, inducing a better separation (S) capability. The scatter plot shows the event distribution of dX4 (X = A, C, T), which all can be discriminated by K238Q.

Results

Assessment of the New K238Q Aerolysin Nanopore

We examined the sensing ability of K238Q with our model segment, dA4. The residence time of the interaction is taken as an indicator which demonstrated two orders of magnitude increase than the WT, reached 122 ± 19 ms at 120 mV bias voltage. The prolonged residence time reflects that the dA4 and nanopore interaction is enhanced attributes to the glutamine (Q) replacement. The residue current (Ires) of the interaction is also regarded. In the case of dA4 probed by K238Q, the Ires is ca. 34.5 ± 0.5 pA (corresponds to Ires/I0 = 0.58) which is only slightly higher than WT (Ires/I0 = 0.51). Through the test with the model segment, the strong interaction between Glutamine (Q) and the nucleotide base reflects in the much longer residence time. The performance of K238Q discriminating dA4 with small half-peak width (1.0 pA) suggesting good nucleotide identifying ability with great potential.

Then, other homo-oligonucleotide, deoxycytosine dC4, and deoxythymine dT4 were added successively to the dA4 measurement. Each substrate appeared as a distinguishable signature signal, which suggests the single K238Q aerolysin nanopore is capable of identifying the dX4 (X = A, C, T) from the mixed solution. The histogram of the residue currently show a clear Gaussian distribution of the mixture of the dX4. Moreover, considering the peak distribution of Ires, the adenine contributes the smallest value (34.5 ± 0.5 pA), then cytosine (40.1 ± 1.0 pA) and thymine (42.5 ± 1.0 pA). The larger residue current corresponds to a larger steric exclusion. Comparing the molecular weight of the four nucleoside bases (G < A < T < C), the steric effect of single nanopore analysis however is in a different order (A < G < T < C). The result suggests the noncovalent interaction is more pronounced than the steric effect interaction. From a structure inspection, the replacement of the positively charged arginine (K) with uncharged glutamine (Q) makes the lumen more negatively charged due to the unpairing with the neighboring E258 causing a decreased energy barrier for the cations. Moreover, the glutamine interact strongly with nucleotide base attributes to hydrogen bond as well as the van der Waals force.29 Typically, glutamine (Q) interacts strongest with adenine, which in turn explains the best identification to our model segment, dA4. Moreover, dA4, dC4, and dT4 have a separation (S) of SdA-dT = 3.67 pA and SdT-dC = 1.23 pA. The strongest noncovalent interaction between Q and dA provides the best separation ability of K238Q in discriminating the homonucleotide segments.

Figure 2.

Figure 2

Open in a new tab

Distribution of current and duration from the K238Q interaction with X4 (X = A, C, T). The color bar illustrates the accumulation of each DNA segment. From the top figure, A4 was first introduced, and then C4 and T4 were added successively with different peaks that correlate to each segment. A histogram of the current distribution of the mixture was plotted in the bottom figure to demonstrate the current distribution and separation ability of K238Q. The data were acquired in 1.0 M KCl, 10 mM Tris, and 1.0 mM EDTA at pH 8.0 and +120 mV in the presence of 2.0 μM dX4.

Single Base Detection

WT aerolysin could discriminate homonucleotide segments, 5′-XA3 (X = A, G, T, C), in a mixture solution,30 while the discrimination of heteronucleotides remains poor due to insufficient current resolution. The K238Q aerolysin nanopore is evaluated to probe a single nucleotide base in a heteronucleotide sequence. We then designed the heteronucleotide as 5′-XGTA (X = A, C, T, G). The heteronucleotide segments were mixed sequentially and examined by K238Q, and the results are given in Figure 3. The two current indicators, residue current and residence time of the blockages, are examined. Regarding to residue current, the blockages show a clear Ires, which is centered at 44.40 ± 0.22 pA, 46.16 ± 0.24 pA, and 46.78 ± 0.40 pA for AGTA, TGTA, and CGTA respectively. The averaged residence time for AGTA has the largest duration, which is 49.42 ms, and TGTA and CGTA have a duration of 22.63 and 11.08 ms, respectively. The current separation of the analytes is SAGTA-TGTA = 1.76 pA and STGTA-CGTA = 0.62 pA.

Figure 3.

Figure 3

Open in a new tab

Distribution of current and duration from K238Q interaction with XGTA (X = A, C, T). The color bar illustrates the accumulation of each DNA segment. From the top figure, AGTA was first introduced, and then TGTA and CGTA were added successively with different peaks that correlate to each segment. A histogram of the current distribution of the mixture was plotted at the bottom figure to demonstrate the current distribution and separation ability of K238Q. The data were acquired in 1.0 M KCl, 10 mM Tris, and 1.0 mM EDTA at pH 8.0 and +140 mV in the presence of 2.0 μM XGTA.

To this end, we have shown that the K238Q could discriminate a single base difference in a heteronucleotide mixture without labeling or an amplification process.

Single Base Modification Detection

The WT aerolysin has shown its capability to discriminate the single methylation group at the 5′ end of homonucleotide segments (i.e., CAAA versus mCAAA).18 Later, a mutant analogue K238G31 enables the discrimination of methylation that occurred in the second position from the 5′ end of a randomly designed heteronucleotide segment (i.e., ACGA vs AmCGA). However, other types of epigenetic modifications could not be properly discriminated due to poor sensitivity. We herein evaluate the amperometric resolution of K238Q in sensing the three types of DNA lesions (i.e., oG, I, mC) occurring in a heteronucleotide segment.

The nucleotide, 5′-ACGA, as the model segment, is directly read by the K238Q. As examples of epigenetic modification, the guanine (G) was modified into I or oG, respectively. Moreover, methylation was introduced into the cytosine (C). The modified model nucleotides are denoted as 5′-ACIA, 5′-ACoGA, and 5′-AmCGA. As expected, the K238Q could discriminate each type of lesion from each other as demonstrated in Figure 3a. Following the mass increase order of 5′-ACIA, 5′-ACGA, 5′-AmCGA, 5′-ACoGA, the histogram of the residue current was centered at 59.32 ± 0.44 pA, 58.61 ± 0.42 pA, 57.28 ± 0.35 pA, and 60.30 ± 0.49 pA respectively. The residence times are averaged at 11.37, 15.14, 30.13, and 12.07 ms. Neither the current nor the residence time matches the order of mass magnitude; moreover, the modification on the single nucleotide base makes ca. 20 Da difference in molecule weight, which is less than 2% of the model nucleotide. It is assumed that the separation of different types of epigenetic modification is achieved predominantly from the noncovalent interaction between nucleotide base and the sensing region rather than simply the steric effect which is negligible. Regarding the separation capability of K238Q in lesion detection, SAmCGA-ACGA = 1.33 pA, SACGA-ACIA = 0.71 pA, and SACIA-ACoGA = 0.98 pA, which are much better than the K238G (data not shown). However, the TGTA and GGTA could not be discriminated by this nanopore even under strong osmotic pressure (Figure S4), which needs further improvements. In order to prove that the superior separation capability comes from the noncovalent interaction, the methylcytosine (mC) at different positions on a nucleotide segments is designed and examined by the K238Q.

Following the previous model segment, 5′-ACGA, another CG pair is introduced making a longer model nucleotide 5′-ACGCGA. The cytosine at both second and fourth positions are methylated. Since the weight of the methylation group is negligible and the nucleotide translocates linearly across the sensing region, the longer model nucleotide is modified into 5′-AmCGCGA and 5′-AmCGmCGA. The mixture is probed by K238Q, and the residue current as well as residence time are adopted to analyze the blockages. The results are given inFigure 5 showing the powerful capability of K238Q in discriminating methylcytosine at different positions of a nucleotide segment. Such outcome could not be achieved by the other aerolysin nanopores especially under label-free, heterocomposition, and mixed condition.

Figure 5.

Figure 5

Open in a new tab

Distribution of current and duration from K238Q interaction with ACGCGA and the modified DNA segments. The color bar illustrates the accumulation of each DNA segment. From the top figure, AmCGmCGA was first introduced, and then AmCGCGA and ACGCGA were added successively with different peaks raised correlate to each segment. A histogram of the current distribution of the mixture was plotted in the bottom panel to demonstrate the current distribution and separation ability of K238Q. The data were acquired in 1.0 M KCl, 10 mM Tris, and 1.0 mM EDTA at pH 8.0 and +140 mV in the presence of 2.0 μM ACGCGA and the modified segments.

Figure 4.

Figure 4

Open in a new tab

Distribution of current and duration from K238Q interaction with ACGA and the modified segments. The color bar illustrates the accumulation of each DNA segment. From the top figure, AmCGA was first introduced, and then ACGA, ACIA, and ACoGA were added successively with different peaks that correlate to each segment. A histogram of the current distribution of the mixture was plotted in the panel to demonstrate the current distribution and separation ability of K238Q. The data were acquired in 1.0 M KCl, 10 mM Tris, and 1.0 mM EDTA at pH 8.0 and +180 mV in the presence of 2.0 μM ACGA and the modified segments.

It is noteworthy that the previous 5′-ACGA model showed the mass contribution is negligible, and the trend matches well with the noncovalent interaction from different position. More specifically, the outcome demonstrated that the current distribution of detected ACGCGA, AmCGCGA, and AmCGmCGA are centered at 36.36 ± 0.58 pA, 35.36 ± 0.52 pA, and 33.42 ± 0.49 pA respectively. The residence times are averaged 39.7, 26.1, and 21.8 ms, respectively. Regarding the separation ability of K238Q to the modified base at different positions, SAmCGmCGA-AmCGCGA = 1.36 pA and SAmCGCGA-ACGCGA = 0.61 pA. On the basis of the previous oligonucleotide interaction with K238Q, the presence of adenine interacts strongly with Q, which provides the longest residence time, while cytosine interacts the weakest with Q and provides a relatively short residence time. Moreover, the nucleotide containing adenine gives the largest separation than the other segments. According to this correlation, we could assume that the glutamine (Q) interacts more strongly with methylcytosine (mC) than cytosine (dC). Even the mC position and quantification with glutamine (Q) could be measured or probed in the aerolysin nanopore lumen with the specified design.

Conclusion

The K238Q aerolysin nanopore achieved discrimination of different types of epigenetic modified nucleotide at different positions with no labeling and amplification. The point mutation K238Q prolongs the nucleotide residence inside the nanopore lumen, thus enhancing the noncovalent interaction to be distinguishable.

We first probed homonucleotide, and taking the example of dA4, the residence time is prolonged two orders of magnitude than WT. More importantly, neither of the two important parameters, residue current and residence time, showed a correlation with the mass contribution, or steric effect. To prove this, we designed a heteronucleotide with a single base and even side chain difference. The K238Q well separated the mixture of these samples, again, not following the steric trend. To this point, the epigenetic modifications contribute less than 2% of the weight, and consequently, we assume the mass contribution is not dominant in our nanopore analysis and could be neglected. Then, a longer model nucleotide with methylated cytosine at different positions is introduced for the purpose of single nucleotide base precise probing. Both the current and residence time decrease with the increase in the number of methylation sites at different positions. We finally could conclude that the noncovalent interaction is sensed and utilized for the separation of different types of epigenetic modifications at different positions.

Taking advantage of the superior sensing ability for the epigenetic modification by K238Q, the application could be applied to real-time monitoring the enzyme cleavage process,8 even achieving quantification of different types of damaged nucleotide bases. The analysis of a longer nucleotide chain could be obtained by optimizing the measuring condition (e.g., LiCl, MgCl2).32 Better current separation (S) could be achieved by applying an asymmetric salt concentration gradient. By controlling the amino acid composition at the sensing region, nucleotide bases and modified bases interaction with amino acid could be predicted, and even the interaction between amino acid groups from a peptide could be mapped, due to its confined space.33−36 Moreover, the parallelization and miniaturization of the nanopore based single molecule label-free detection method could be applied to real sample diagnoses, with the most prominent example being the recognition of a conformation change of a homonucleotide with its application.37,38

Materials and Methods

All model nucleotides and modified nucleotides were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Chemical compounds such as KCl (≥99%) and decane (anhydrous, ≥99%) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Phosphor lipid (1,2-diphytanoyl-snglycero-3-phosphocholine) (powder, ≥99%) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The other chemical compounds were obtained from Aladine (Shanghai, China). Proaerolysin was obtained from Escherichia coli according our previous reports.21,22 For experimental and characterization detail, please refer to Supporting Information. The membrane formation and single nanopore experiments were performed identical to the standard protocol.39 Single channel current was obtained from a patch clamp amplifier (Axon 200B) and digitized by an A/D converter (DigiData 1440A), Molecular Devices, Sunnyvale, CA, USA. All the data points were sampled at 100 kHz and filtered at 5 kHz. The data analysis procedure follows the previous work.30

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.9b01129.

  • Detailed K238Q purification process and single-channel electrochemical methodologies. Figure S1 shows the sequencing spectra of the K238Q plasmid. Figure S2 shows the SDS page characterization of K238Q and WT aerolysin. Figure S3 gives the I–V behavior of K238Q and WT aerolysin. Figure S4 shows discrimination of XGTA by K238Q under asymmetry conditions (PDF)

Author Contributions

# J.W., M.-Y. L., and J. Y. contributed equally. Y-L.Y. and Y-T.L. initiated the project. J.W., M-Y.L., and J.Y. performed the experiments and data analysis. Y-Q.W., X-Y.W., and J. H. mutated the protein. J. W., M-Y. L., J. Y., Y-L. Y., and Y-T. L. were involved in the manuscript writing. The manuscript was written through contributions of all authors.

This research was supported by National Natural Science Foundation of China (21834001 and 61871183, 61901171). Y.-L.Y. is sponsored by National Ten Thousand Talent Program for Young Top-Notch Talent. J.W. is sponsored by the Shanghai Sailing Program (19YF1410500) and China Postdoctoral Science Foundation (2019M651412, 2019T120309).

The authors declare no competing financial interest.

Supplementary Material

oc9b01129_si_001.pdf (2.4MB, pdf)

References

  1. Cadet J.; Poulsen H. Measurement of oxidatively generated base damage in cellular DNA and urine. Free Radical Biol. Med. 2010, 48 (11), 1457. 10.1016/j.freeradbiomed.2010.03.004. [DOI] [PubMed] [Google Scholar]
  2. Wu H.; Zhang Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 2014, 156 (1–2), 45. 10.1016/j.cell.2013.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kasianowicz J. J.; Brandin E.; Branton D.; Deamer D. W. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770–13773. 10.1073/pnas.93.24.13770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Lieberman K. R.; Cherf G. M.; Doody M. J.; Olasagasti F.; Kolodji Y.; Akeson M. Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J. Am. Chem. Soc. 2010, 132 (50), 17961. 10.1021/ja1087612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Rincon-Restrepo M.; Mikhailova E.; Bayley H.; Maglia G. Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes. Nano Lett. 2011, 11 (2), 746. 10.1021/nl1038874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Butler T. Z.; Pavlenok M.; Derrington I. M.; Niederweis M.; Gundlach J. H. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (52), 20647. 10.1073/pnas.0807514106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Soskine M.; Biesemans A.; Moeyaert B.; Cheley S.; Bayley H.; Maglia G. An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry. Nano Lett. 2012, 12 (9), 4895. 10.1021/nl3024438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goyal P.; Krasteva P. V.; Van Gerven N.; Gubellini F.; Van den Broeck I.; Troupiotis-Tsailaki A.; Jonckheere W.; Pehau-Arnaudet G.; Pinkner J. S.; Chapman M. R.; Hultgren S. J.; Howorka S.; Fronzes R.; Remaut H. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 2014, 516 (7530), 250. 10.1038/nature13768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cao C.; Ying Y.-L.; Hu Z.-L.; Liao D.-F.; Tian H.; Long Y.-T. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore. Nat. Nanotechnol. 2016, 11 (8), 713. 10.1038/nnano.2016.66. [DOI] [PubMed] [Google Scholar]
  10. Bräuning B.; Bertosin E.; Praetorius F.; Ihling C.; Schatt A.; Adler A.; Richter K.; Sinz A.; Dietz H.; Groll M. Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB. Nat. Commun. 2018, 9 (1), 1806. 10.1038/s41467-018-04139-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang G.; Voet A.; Maglia G. FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 Da resolution. Nat. Commun. 2019, 10 (1), 835. 10.1038/s41467-019-08761-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wang J.; Bafna J.; Bhamidimarri S.-P.; Winterhalter M. Small molecule permeation across membrane channels: Chemical modification to quantify transport across OmpF. Angew. Chem., Int. Ed. 2019, 58, 4737. 10.1002/anie.201814489. [DOI] [PubMed] [Google Scholar]
  13. Podobnik M.; Savory P.; Rojko N.; Kisovec M.; Wood N.; Hambley R.; Pugh J.; Wallace E. J.; McNeill L.; Bruce M.; Liko I.; Allison T. M.; Mehmood S.; Yilmaz N.; Kobayashi T.; Gilbert R. J. C.; Robinson C. V.; Jayasinghe L.; Anderluh G. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat. Commun. 2016, 7, 11598. 10.1038/ncomms11598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. van den Berg B.; Bhamidimarri S. P.; Prajapati J. D.; Kleinekathöfer U.; Winterhalter M. Outer-membrane translocation of bulky small molecules by passive diffusion. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (23), E2991. 10.1073/pnas.1424835112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Locher K. P.; Rees B.; Koebnik R.; Mitschler A.; Moulinier L.; Rosenbusch J. P.; Moras D. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 1998, 95 (6), 771. 10.1016/S0092-8674(00)81700-6. [DOI] [PubMed] [Google Scholar]
  16. Schibel A. E. P.; An N.; Jin Q.; Fleming A. M.; Burrows C. J.; White H. S. Nanopore detection of 8-Oxo-7,8-dihydro-2 ′-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site. J. Am. Chem. Soc. 2010, 132 (51), 17992. 10.1021/ja109501x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jin Q.; Fleming A. M.; Johnson R. P.; Ding Y.; Burrows C. J.; White H. S. Base-excision repair activity of uracil-DNA glycosylase monitored using the latch zone of alpha-Hemolysin. J. Am. Chem. Soc. 2013, 135 (51), 19347. 10.1021/ja410615d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iacovache I.; De Carlo S.; Cirauqui N.; Dal Peraro M.; van der Goot F. G.; Zuber B. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nat. Commun. 2016, 7, 12062. 10.1038/ncomms12062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yu J.; Cao C.; Long Y. T. Selective and sensitive detection of methylcytosine by Aerolysin nanopore under serum condition. Anal. Chem. 2017, 89 (21), 11685. 10.1021/acs.analchem.7b03133. [DOI] [PubMed] [Google Scholar]
  20. Cao C.; Li M.-Y.; Cirauqui N.; Wang Y.-Q.; Dal Peraro M.; Tian H.; Long Y.-T. Mapping the sensing spots of aerolysin for single oligonucleotides analysis. Nat. Commun. 2018, 9 (1), 2823. 10.1038/s41467-018-05108-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ying Y.-L.; Long Y.-T. Nanopore-based single-biomolecule interfaces: From information to knowledge. J. Am. Chem. Soc. 2019, 141 (40), 15720. 10.1021/jacs.8b11970. [DOI] [PubMed] [Google Scholar]
  22. Wang Y..-Q.; Cao C.; Ying Y.-L.; Li S.; Wang M.; Huang J.; Long Y.-T. Rationally designed sensing selectivity and sensitivity of an aerolysin nanopore via site-directed mutagenesis. ACS Sens. 2018, 3 (4), 779. 10.1021/acssensors.8b00021. [DOI] [PubMed] [Google Scholar]
  23. Wang Y.-Q.; Li M.-Y.; Qiu H.; Cao C.; Wang M.-B.; Wu X.-Y.; Huang J.; Ying Y.-L.; Long Y.-T. Identification of essential sensitive regions of the aerolysin nanopore for single oligonucleotide analysis. Anal. Chem. 2018, 90 (13), 7790. 10.1021/acs.analchem.8b01473. [DOI] [PubMed] [Google Scholar]
  24. Wu X.-Y.; Wang M.-B.; Wang Y.-Q.; Li M.-Y.; Ying Y.-L.; Huang J.; Long Y.-T. Precise construction and tuning of an aerolysin single-biomolecule interface for single molecule sensing. Chin. Chem. Soc. 2019, 1 (3), 304. [Google Scholar]
  25. Li M.-Y.; Wang Y.-Q.; Lu Y.; Ying Y.-L.; Long Y. Single molecule study of hydrogen bond interactions between single oligonucleotide and aerolysin sensing interface. Front. Chem. 2019, 7, 528. 10.3389/fchem.2019.00528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lu Y.; Wu X.-Y.; Ying Y.-L.; Long Y.-T. Simultaneous single-molecule discrimination of cysteine and homocysteine with a protein nanopore. Chem. Commun. 2019, 55 (63), 9311. 10.1039/C9CC04077C. [DOI] [PubMed] [Google Scholar]
  27. Wang J.; Li M.-Y.; Yang J.; Wu X.-Y.; Huang J.; Ying Y.-L.; Long Y.-T. Label-free detection of solo oligonucleotide lesion based on site-direct mutagenized aerolysin nanopore. Biophys. J. 2019, 116 (3), 148a. 10.1016/j.bpj.2018.11.822. [DOI] [Google Scholar]
  28. Li M.-Y.; Wang Y.-Q.; Ying Y.-L.; Long Y.-T. Revealing the transient conformations of a single flavin adenine dinucleotide using an aerolysin nanopore. Chem. Sci. 2019, 10, 10400. 10.1039/C9SC03163D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hu Z. L.; Li Z. Y.; Ying Y. L.; Zhang J.; Cao C.; Long Y. T.; Tian H. Real-time and accurate identification of single oligonucleotide photoisomers via an aerolysin nanopore. Anal. Chem. 2018, 90 (7), 4268. 10.1021/acs.analchem.8b00096. [DOI] [PubMed] [Google Scholar]
  30. Luscombe N. M.; Laskowski R. A.; Thornton J. M. Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Res. 2001, 29 (13), 2860. 10.1093/nar/29.13.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Cao C.; Yu J.; Li M.-Y.; Wang Y.-Q.; Tian H.; Long Y.-T. Direct readout of single nucleobase variations in an oligonucleotide. Small 2017, 13 (44), 1702011. 10.1002/smll.201702011. [DOI] [PubMed] [Google Scholar]
  32. Wang Y. Q.; Li M. Y.; Qiu H.; Cao C.; Wang M. B.; Wu X. Y.; Huang J.; Ying Y. L.; Long Y. T. Identification of essential sensitive regions of the aerolysin nanopore for single oligonucleotide analysis. Anal. Chem. 2018, 90 (13), 7790. 10.1021/acs.analchem.8b01473. [DOI] [PubMed] [Google Scholar]
  33. Hu Z.-L.; Li M.-Y.; Liu S.-C.; Ying Y.-L.; Long Y.-T. A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA. Chem. Sci. 2019, 10 (2), 354. 10.1039/C8SC03927E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ying Y.-L.; Zhang J.; Gao R.; Long Y.-T. Nanopore-based sequencing and detection of nucleic acids. Angew. Chem., Int. Ed. 2013, 52 (50), 13154. 10.1002/anie.201303529. [DOI] [PubMed] [Google Scholar]
  35. Ying Y.-L.; Cao C.; Hu Y.-X.; Long Y.-T. A single biomolecule interface for advancing the sensitivity, selectivity and accuracy of sensors. Nat. Sci. Rev. 2018, 5 (4), 450. 10.1093/nsr/nwy029. [DOI] [Google Scholar]
  36. Wang J.; Yang J.; Ying Y.-L.; Long Y.-T. Nanopore-based confined spaces for single-molecular analysis. Chem. - Asian J. 2019, 14 (3), 389. 10.1002/asia.201801648. [DOI] [PubMed] [Google Scholar]
  37. Gu Z.; Ying Y.-L.; Long Y.-T. Nanopore sensing system for high-throughput single molecular analysis. Sci. China: Chem. 2018, 61 (12), 1483. 10.1007/s11426-018-9312-3. [DOI] [Google Scholar]
  38. Guo J.; Chen Y.; Jiang Y.; Ju H. Polyadenine-modulated dna conformation monitored by surface-enhanced raman scattering (SERS) on multibranched gold nanoparticles and its sensing application. Chem. - Eur. J. 2017, 23 (39), 9332. 10.1002/chem.201700883. [DOI] [PubMed] [Google Scholar]
  39. Abbassi-Daloii T.; Yousefi S.; de Klerk E.; Grossouw L.; Riaz M.; t Hoen P. A. C.; Raz V. An alanine expanded PABPN1 causes increased utilization of intronic polyadenylation sites. NPJ. Aging and Mechanisms of Disease 2017, 3, 6. 10.1038/s41514-017-0007-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Cao C.; Liao D.-F.; Yu J.; Tian H.; Long Y.-T. Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis. Nat. Protoc. 2017, 12 (9), 1901. 10.1038/nprot.2017.077. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

oc9b01129_si_001.pdf (2.4MB, pdf)

Articles from ACS Central Science are provided here courtesy of American Chemical Society

RESOURCES