Supporting Information

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1 Supporting Information A Universal Strategy for Aptamer-Based Nanopore Sensing through Host-Guest Interactions inside a-hemolysin** Ting Li, Lei Liu, Yuru Li, Jiani Xie, and Hai-Chen Wu* anie_ _sm_miscellaneous_information.pdf

2 Contents S1. Materials and Methods S2 S2. DNA modification and characterization S6 S3. Translocation of DNA1-Ad βcd through αhl S8 S4. Control experiments S9 S5. Assignment of the current Level 1 in signature events S10 S6. Assignment of the current Level 2 in signature events S11 S7. Assignment of the current Level M in signature events S14 S8. Competing experiment with 1-aminoadamantane S16 S9. Detection of thrombin S17 S10. Detection of cocaine S19 S11. Influence of the addition of magnetic beads on events S21 S12. Supporting Information Tables S22 S13. Supporting Information References S24 S1

3 S1. Materials and Methods Materials and characterization. All the DNA samples were purified by HPLC and purchased from Sangon Biotechnology Co., Ltd. (Shanghai). 1,2-Diphytanoyl-sn-glycero-3-phosphochline (DPhPc) was purchased from Avanti Polar Lipids (Alabaster, AL). All the chemicals were purchased from Sigma-Aldrich, Alfa Aesar and J&K, and used without further purification. Cocaine hydrochloride was bought from National Institute for the Control of Pharmaceutical and Biological Products (Changchun, China). Recombinant Human VEGF 121 (aa ), Carrier Free (abbreviated as VEGF 121, Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain) was obtained from R&D systems (Minneapolis, MN). Dynabeads MyOne Streptavidin T1 (10mg/mL, beads/ml; beads diameter: 1.0 μm, supplied in PBS; ph 7.4/0.1%BSA/0.02% sodium azide) was obtained from Invitrogen (Shanghai). Human thrombin with a purity of 97.9% was obtained from Haematologic Technologies, Inc. (Essex Junction, VT). BSA (bovine serum albumin) and lysozyme were obtained from Sigma-Aldrich. Micro Bio-Spin P6 gel columns (Tris buffer) were purchased from Bio-Rad (Hercules, CA). The columns were pre-equilibrated three times with 80 μl deionized water prior to use. All the samples and buffers were prepared in deionized water (Millipore, MA). DNA mass spectrometry was analyzed on Thermo-Finnigan LCQ Deca XP Plus. NMR spectra were recorded on Bruker Avance III 500 spectrometer. FT-IR measurements were performed on a Bruker Tensor27 spectrometer. DNA concentration was measured on ND-2000c Spectrophotometer. Protein preparation. Wild type αhl-d8h6 and mutant monomers (K8L, K147N, M113R, M113H, wildtype background with D8H6 tail) were produced by expression in BL21 (DE3) plyss Escherichia coli cells. The monomers were then assembled into homoheptamers on rabbit red blood cell membranes followed by purification with 8% SDS-PAGE as described earlier. [1,2] The purified heptamer protein was conserved in buffer (10 mm Tris-HCl, ph 7.9, 50 mm NaCl) and stored at -70 C. All the mutants used in this work are homoheptamers which means the mutations appear in all the 7 subunits. S2

4 Buffer preparation g of KCl (99.999%, Sigma-Aldrich) and g of Tris HCl (99.0%,Sigma-Aldrich)were dissolved in 80 ml of deionized water (Millipore, MA). 2 M NaOH was used to adjust the ph to 8.0. The solution was diluted with deionized water to 100 ml. The final buffer solution consists of 3 M KCl and 10 mm Tris at ph 8.0. For VEGF 121 and thrombin detection experiments, ph was adjusted to 7.2. General procedure for the preparation of DNA probes. 3.3 μl alkyne-containing DNA (100 μm), 1.2 μl deionized water, 2 μl azidomethylferrocene (dissolved in acetonitrile, 200 mm), 1 μl sodium ascorbate (20 mm), 0.5 μl copper nitrate (20 mm) were added to 2 μl HEPES (100 mm) buffer, with a final volume of 10 μl. The reaction was incubated for 2 hours at room temperature, and then 2 μl EDTA solution (100 mm) was added to terminate the reaction. The DNA product was purified with Micro Bio-spin P6 columns. Next, 10 μl CB[7] aqueous solution (5.0 mm) was added to the DNA solution and incubated for 2 hours to afford the final DNA probes. VEGF 121 detection experiments. The VEGF 121 probe DNA3 was first modified with azidomethylferrocene to afford DNA3-Fc following the above procedure. Then, DNA3-Fc (8.2 μl 32.4 μm) was annealed with VEGF 121 binding aptamer DNA2 (2 μl, 132 μm) in 9.8 μl annealing buffer (150 mm NaCl, 10 mm Tris, ph 7.4). The total volume was 20 μl. After annealing, 10 μl CB[7] (5 mm) was added and incubated for 2 h. Next, the DNA duplex was incubated with various concentrations of VEGF 121 (final concentration: 0.5, 5, 20, 50, 100, 200 nm) or BSA (10 μm), lysozyme (10 μm) and thrombin (200 nm) respectively for 2 h at 37 C. Binding buffer (100 mm NaCl, 5 mm KCl, 10 mm Tris-HCl, ph 7.4) was added to make the final solution volume 50 μl before incubation. The resulting solution was subject to single channel recording experiments. Thrombin detection experiments. The thrombin probe DNA5 which bears a terminal alkyne group was first modified with azidomethylferrocene following the general procedure to afford DNA5-Fc. Then, DNA5-Fc (8.3 μl, 33.7 μm) was annealed with TBA-containing DNA4 (2.0 μl, 14.1 μm) in 9.7 μl buffer of 150 mm NaCl, 10 mm Tris, ph 7.4. After annealing, 10 μl CB[7] aqueous solution (5.0 mm) was added to the solution and kept for 2 hours. Next, the S3

5 DNA duplex was incubated with various concentrations of thrombin (final concentration: 5, 20, 50, 100, 200, 300 nm) or BSA (10 μm) or lysozyme (10 μm) in the buffer of 140 mm NaCl, 5.0 mm KCl, 5.0 mm MgCl 2, 20 mm Tris-HCl, ph 7.4 for overnight at 37 C (total volume 50 μl). The obtained solution was ready for single-channel recording experiments. Cocaine detection experiments. The cocaine probe DNA7 was first modified with azidomethylferrocene to afford DNA7-Fc following the general procedure. Then, DNA7-Fc (7.9 μl, 34.3 μm) was annealed with cocaine binding aptamer DNA6 (2 μl, 135 μm) in 10.1 μl annealing buffer (150 mm NaCl, 10 mm Tris, ph 7.4). The total volume was 20 μl. After annealing, 10 μl CB[7] (5 mm) was added and incubated for 2 h. Next, the DNA duplex was incubated with various concentrations of cocaine (final concentration: 5, 50, 100, 300, 500, 1000 μm) for 2 h at 37 C. Binding buffer (100 mm NaCl, 5 mm KCl, 10 mm Tris-HCl, ph 7.4) was added to make the final solution volume 50 μl before incubation. The resulting solution was subject to single channel recording experiments. Improved detection of VEGF 121 by using magnetic beads. VEGF 121 probe DNA3-Fc was obtained as described above. Then DNA3-Fc (8.0 μl, 32.4 μm) was annealed with 3-biotinated VEGF 121 binding aptamer DNA8 (2.0 μl, 129 μm) in 10 μl buffer of 150 mm NaCl, 10 mm Tris, ph 7.4. After annealing, the DNA duplex was incubated with 10 μl CB[7] aqueous solution (5.0 mm) for 2 h. Meanwhile, MBs suspension (50 μl, 10 mg/ml) was washed 3 times with 1 ml 1 BW buffer (2 M NaCl, 1 mm EDTA, 10 mm Tris-HCl, ph 7.5). Next, the DNA duplex hybrid was mixed with MBs in 200 μl 1 BW buffer, and vortexed for 15 min. The suspension was decanted after MBs were concentrated with a magnet. The MBs were washed 5 times with 0.5 ml 1 BW buffer. Various concentrations of VEGF 121 (final concentration: 500, 300, 100, 50, 5 pm) was incubated with MBs in 1 ml binding buffer (TBS: 10 mm Tris-HCl, 100 mm NaCl, 5 mm KCl, ph 7.4) at 37 C for 2 h with occasional vortexing. After incubation, the supernatant was collected with the aid of a magnet and MBs were washed with deionized water. The solutions were combined and ultra-centrifuged using Amicon Ultra-0.5 centrifugal Filter (3 KD). The concentrated sample was ready for single channel recording experiments. S4

6 Single-channel current recording. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine was used to form a synthetic lipid bilayer across an aperture μm in diameter in a 25-μm-thick polytetrafluoroethylene film (Goodfellow, Malvern, PA) that divided a planar bilayer chamber into two compartments, cis and trans. Both compartments contained 1 ml of buffer solution. DNA samples were added to the cis compartment, which was connected to ground. The trans compartment was connected to the head-stage of the amplifier. All experiments were carried out in 3 M KCl, 10 mm Tris, ph 8.0, at 22.5 ± 2 C, unless otherwise stated. Ionic currents were measured by using Ag/AgCl electrodes with a patch-clamp amplifier (Axopatch 200B; Axon instruments, Foster City, CA), filtered with a low-pass Bessel filter with a corner frequency of 10 khz and then digitized with a Digidata 1440A A/D converter (Axon Instruments) at a sampling frequency of 100 khz. Data analysis. Current traces were analyzed with Clampfit 10.2 software (Axon Instruments). Events were detected using the Event Detection feature, and used to construct amplitude and dwell time histograms. Origin (Microcal, Northampton, MA) and Clampfit were used for histogram construction, curve fitting and graph presentation. Adobe Illustrator was used for making figures. Current signature events were manually selected for statistical analysis. S5

7 S2. DNA modification and characterization Figure S1. Modification of DNA1 with azidomethylferrocene or 1-azidoadamantane and mass spectrometry characterization. (a) Chemical modification of DNA1 with azidomethylferrocene via click chemistry. (b) Chemical modification of DNA1 with 1-azidoadamantane via click chemistry. (c) Mass spectrometry characterization of DNA1. (d) Mass spectrometry characterization of the product DNA1-Fc. (e) Mass spectrometry characterization of the product DNA1-Ad. S6

8 Figure S2. Modification of the DNA probes with Fc and mass spectrometry characterization. (a) Mass spectrometry characterization of VEGF 121 aptamer DNA3. (b) Mass spectrometry characterization of the product DNA3-Fc. (c) Mass spectrometry characterization of TBA DNA5. (d) Mass spectrometry characterization of the product DNA5-Fc. (e) Mass spectrometry characterization of cocaine aptamer DNA7. (f) Mass spectrometry characterization of the product DNA7-Fc. S7

9 S3. Translocation of DNA1-Ad βcd through αhl Figure S3. Translocation of DNA1-Ad βcd through αhl. (a) A representative current trace of the translocation of DNA1-Ad βcd (final concentration 0.25 μm) through αhl. Data were acquired in the buffer of 3 M KCl and 10 mm Tris, ph 8.0, with the transmembrane potential held at +160 mv. Red arrows indicate the moderately long current events. (b) Expanded view of a typical long and deep blockage current event. (c) The dwell time histogram of the long events. S8

10 S4. Control experiments Figure S4. Translocation traces of control experiments. (a) DNA1. (b) Normal DNA. The sequence of the normal DNA is the same as that of DNA1 except that there is no modification on the middle T base. (c) CB[7] in cis (concentration: 50 μm). (d) DNA1 and CB[7] (concentration: 50 μm). (e) DNA1-Fc. All the DNA concentrations were 1.0 μm. DNA and CB[7] were all placed in cis. Data were acquired in the buffer of 3 M KCl and 10 mm Tris, ph 8.0, with the transmembrane potential held at +160 mv. S9

11 S5. Assignment of the current Level 1 in signature events Since none of the control experiments showed any prolonged deep current blockades (for control experimental traces, see Figure S4), we attributed the Level 1 state to the translocation of DNA1-Fc CB[7] and concomitant dissociation of the Fc CB[7] complex at the constriction of αhl. Two features are supportive of this assignment. First, high voltage was required to generate characteristic events. When the transmembrane potential was lower than 140 mv, we only observed very few signature events. This might be because the tight binding of Fc CB[7] requires a strong electric field to provide sufficient driving force on DNA molecules to unbind the DNA1-Fc CB[7] hybrid. The trend of the frequency of signature events under different applied voltages also supports the conclusion (Figure S5). Second, the Level 1 state appeared in both translocation traces of DNA1-Ad βcd and DNA1-Fc CB[7], and the difference between the mean duration of this level (37.7 ms versus ms) correlates well with the binding constants of Ad βcd and Fc CB[7] complexes ( M -1 versus M -1 ). [3,4] Figure S5. Voltage-dependence of the frequency of signature events. As the applied potential increases from 160 mv to 240 mv, the frequency of multi-level signature events rose exponentially. This clearly indicates that higher transmembrane potential facilitates the unbinding of the Fc CB[7] complex and also promotes DNA translocation from cis to trans. Traces were recorded in 3 M KCl buffered with 10 mm Tris, ph 8.0. DNA1-Fc (final concentration: 0.25 μm) were incubated with CB[7] (final concentration 50 μm) at room temperature for 2 hours before measurement. S10

12 S6. Assignment of the current Level 2 in signature events It is interesting that the Level 2-2 and Level M states only existed in the translocation trace of DNA1-Fc CB[7], but not in that of DNA1-Ad βcd. Therefore, these three states are strongly associated with the existence of CB[7]. The current oscillation between Levels 2 and 2 is reminiscent of trapping an analyte in the nanocavity of a protein pore. [5] Thus, we speculated that the Level 2-2 alternation was due to the trapping and oscillation of CB[7] inside the vestibule of αhl. It is well known that cucurbiturils have strong affinity with positively charged organic guests. [6] We presumed that the side chains of some basic amino acids inside the vestibule might have interactions with CB[7] so as to cause the current oscillation. By checking through the sequence of the vestibule region, we found that the two lysine (K) residues at positions 8 and 147 might be crucial for the temporal trapping of CB[7] in the vestibule. While K-8 is at the cis opening of the vestibule and K-147 is near the constriction, the positively charged amino groups on the side chain of lysine might act as the gatekeepers of the nanocavity and thus retain CB[7] for a certain period of time (Figure S6). To validate this assumption, we carried out translocation of DNA1-Fc CB[7] through αhl mutants K147N and K8L, respectively. The results provided compelling evidence that the lysines at position 8 played important roles in the trapping and oscillation of CB[7] inside the vestibule (Figure S7). S11

13 Figure S6. Positions of amino acids K8, K147 and M113 inside αhl. (a) Top view. (b) Side view. The images were generated using PyMOL software based on the crystal structure of αhl. S12

14 Figure S7. Translocation of DNA1-Fc CB[7] through engineered αhl mutants K8L and K147N. (a) Representative current trace of the translocation of DNA1-Fc CB[7] through K8L mutant. The frequency of the multi-level events was only reduced by ~25% in K8L mutant compared with wildtype αhl. The red arrows indicate multi-level current events. (b) Expanded views of two current events of type-i and type-ii respectively. It was shown that in both types of events the Level 1 remains about the same, but the duration of Level 2 was shortened to ~10 ms and the Level 2 completely disappeared. (c) Representative current trace of the translocation of DNA1-Fc CB[7] through mutant K147N. In this mutant, the multi-level signature events completely vanished and the frequency of events dropped to only ~1 s -1. The significantly reduced DNA capture rate was due to the loss of positive charge at the constriction of αhl. Traces were recorded in 3 M KCl buffered with 10 mm Tris, ph 8.0. DNA1-Fc (final concentration: 0.25 μm) were incubated with CB[7] (final concentration 50 μm) at room temperature for 2 hours before measurement. The transmembrane potential was held at +200 mv. S13

15 S7. Assignment of the current Level M in signature events Level M in type-ii events has a current blockade smaller than Level 1 but greater than Level 2 (Figure 2d). Based on the amplitude of this level, we speculated that it was caused by the binding of CB[7] with the constriction part of αhl. Thus, we mutated another key residue M113 in the constriction, which is spatially adjacent to position 147 but in a slightly lower position looking from the cis side, to explore the origin of Level M. It is known in literature that CB[7] has tight binding with arginine (K a, 310 M -1 ) and histidine (K a, 400 M -1 ). [7] When DNA1-Fc CB[7] was tested for the translocation through M113R and M113H mutants, we found that the duration of Level M in type-ii event increased from 31.2 ± 2.2 ms for wildtype αhl to ± ms for M113R and ± 48.5 ms for M113H (Figure S8). These results strongly supported our speculation that Level M was caused by the stochastic binding of CB[7] with amino acid residues in the constriction region of αhl. S14

16 Figure S8. Translocation of DNA1-Fc CB[7] through engineered αhl mutants M113R and M113H. (a) A representative current trace of the translocation of DNA1-Fc CB[7] (final concentration 0.25 μm) through Μ113R mutant. The Level M was significantly prolonged. (b) Dwell-time histogram of Level M in the signature events obtained in M113R mutant. (τ Μ ) M113R = ± ms. (c) A representative current trace of the translocation of DNA1-Fc CB[7] (final concentration 0.25 μm) through Μ113H mutant. The Level M was also significantly prolonged. (d) Dwell-time histogram of Level M in the signature events obtained in M113H mutant. (τ Μ ) M113H = ± 48.5 ms. S15

17 S8. Competing experiment with 1-aminoadamantane Figure S9. Influence of a competing guest molecule on the generation of signature events. When 1-aminoadamantane (final concentration 1.0 μm) was added in cis chamber and incubated with DNA1-Fc CB[7] for 30 min, all the signature events disappeared and only ssdna translocation events were observed. The red arrows indicate characteristic multi-level current events. Traces were recorded in 3 M KCl buffered with 10 mm Tris, ph 8.0. DNA1-Fc (final concentration 0.25 μm) was incubated with CB[7] (final concentration 50 μm) at room temperature for 2 hours before measurement. The transmembrane potential was held at +200 mv. S16

18 S9. Detection of thrombin We also tested the detection of another biomacromolecule thrombin. The size of thrombin is too large to lodge inside the vestibule of αhl pore; therefore direct sensing is not feasible. Rotem et al. developed an approach for thrombin detection by hybridizing the DNA aptamers to a short DNA nucleotide which is covalently attached to a cysteine residue near the cis entrance of αhl. [8] The binding of thrombin to the aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. In our work, after a selection of DNA probes, we hybridized the aptamer-containing DNA4 with Fc CB[7]-modified probe DNA5 (Table S1 and S2). Because thrombin binding aptamer (TBA) only has 15 nucleotides, we added three bases to each end of TBA to ensure a clean background. Addition of thrombin to the solution caused the aptamer-probe duplex to unwind and led to the formation of the cation-stabilized quadruplex. The released probe in the solution generated characteristic current events under applied transmembrane potential (Figure S10a). After a series of concentration-varying experiments, we found that the effective detection range of thrombin is between 0 and 100 nm (Figure S10b). The detection limit of our method is ~5 nm. We also performed the specificity experiments by substituting TBA with lysozyme or BSA. It was shown that the f sig in these two groups was barely higher than the control group. S17

19 Figure S10. Detection of thrombin using the developed method. (a) Schematic illustration of the detection of thrombin with a TBA-containing DNA4 and its complementary DNA5 modified with an Fc CB[7] complex. First, thrombin was mixed with DNA4-DNA5-Fc CB[7] duplex to unwind the duplex by competitive binding with TBA. Then, the freed probe DNA5-Fc CB[7] was translocated through αhl to generate characteristic signature events. We added 3 base pairs to both ends of the aptamer-probe duplex in order to obtain a clean background. (b) Correlation of the frequency of signature events with the concentration of thrombin (final concentration: 300, 200, 100, 50, 20, 5 nm). Number of individual experiments n = 6. (c) Investigation of the selectivity in the detection of thrombin. The final concentration of thrombin is 10 nm, whereas the concentration of BSA and lysozyme is 10 μm. All data were acquired in the buffer of 3 M KCl and 10 mm tris, ph 7.2, with the transmembrane potential held at +160 mv. S18

20 S10. Detection of cocaine Apart from biomacromolecules, we also tested the developed strategy in sensing small organic molecules. Cocaine is an ideal model for testing new analytical techniques, and also there are pressing needs for its rapid and sensitive detection in drug investigations and clinical settings. The detection was carried out following the standard procedure in Figure 1. Cocaine aptamer has 30 nucleotides and a fully complementary probe will make the duplex too stable to unwind, so we designed a 7-nucleotide loop in the aptamer-probe duplex (Table S1 and S2; Figure S11a). The detection was conducted with various concentrations of cocaine. The results showed that the effective detection range of cocaine is between 0 and 500 μm with a detection limit of ~5 μm (Figure S11b). We also examined the possible interference of caffeine on the cocaine detection. The results again confirmed the high specificity of the aptamer-based sensing approach (Figure S11c). S19

21 Figure S11. Detection of cocaine using the developed method. (a) Schematic illustration of the detection of cocaine with a cocaine aptamer DNA6 and a probe DNA7 modified with an Fc CB[7] complex. First, cocaine was mixed with DNA6-DNA7-Fc CB[7] duplex to unwind the duplex by competitive binding with DNA6. Then, the probe DNA7-Fc CB[7] was released and translocated through αhl to generate characteristic signature events. (b) Correlation of the frequency of signature events with the concentration of cocaine (final concentration: 5, 50, 100, 300, 500, 1000 μm). Number of individual experiments n = 3. (c) Investigation of the interference of caffeine with the detection of cocaine. The final concentration of cocaine and caffeine is 500 μm. All data were acquired in the buffer of 3 M KCl and 10 mm tris, ph 7.4, with the transmembrane potential held at +160 mv. S20

22 S11. Influence of the addition of magnetic beads on current events Figure S12. Comparison of the recording traces during the detection of VEGF 121 with and without MBs treatment. (a) A typical current trace recorded during the detection of VEGF 121 following the standard procedure in Figure 1. The dashed rectangle boxes indicate the events of occupation of vestibule by the aptamer-probe duplex; the red arrows indicate signature events. (b) Expanded view of an event that represents the lodging of the aptamer-probe duplex inside the vestibule of αhl. (c) A typical current trace during the detection of VEGF 121 following the MBs treatment procedure. The red arrows indicate signature events. (d) Expanded view of a characteristic current event. The concentration of VEGF 121 is 500 pm in both experiments. All data were acquired in the buffer of 3 M KCl and 10 mm tris, ph 7.2, with the transmembrane potential held at +200 mv. S21

23 S12. Supporting information tables Table S1 Sequences of studied aptamers and probes Name Function Sequence DNA1 Random model 5 -CATATTACACT*CCCACGACTC-3 DNA2 VEGF TGTGGGGGTGGACTGGGTGGGTACC-3 aptamer DNA3 VEGF 121 probe 5 -GGTACCCACCCCT*CCCACCCCCACA-3 DNA4 TBA 5 - TTTGGTTGGTGTGGTTGGTTT-3 DNA5 DNA6 Thrombin probe 5 - AAACCAACCAT*ACCAACCAAA-3 Cocaine aptamer 5 -GACAAGGAAAATCCTTCAATGAAGTGGGTC-3 DNA7 Cocaine probe 5 -GACCCACTTCAT*TTTTCCTTGTC-3 DNA8 Biotinated 5 -TGTGGGGGTGGACTGGGTGGGTACC(A) 10 - VEGF aptamer Biotin-3 Note: Aptamer sequences are highlighted in bold; mismatched bases are underlined; deliberately added bases are in italic. T* alkyne-modified thymine: Biotin + linker: S22

24 Table S2 Sequences tested in designing DNA probes Name Function Sequence and drawback DNA9 VEGF probe 2 DNA10 VEGF probe 3 DNA11 Thrombin probe 2 DNA12 Thrombin probe 3 DNA13 Thrombin probe 4 DNA14 Cocaine probe 2 DNA15 Cocaine probe GGTACCCACCCAT*TCCACCCCCACA -3 one mismatch; the f sig decreases by 50% compared with DNA3 5 - GGTACCCACCACT*CACACCCCCACA -3 five mismatches; multi-level events appear in the control group 5 - CCAACCAT*ACCAACC-3 no added base; the aptamer-probe complex is unstable 5 - ACCAACCAT*ACCAACCA-3 one added base; the aptamer-probe complex is unstable 5 - AACCAACCAT*ACCAACCAA-3 two added bases; the aptamer-probe complex is unstable 5 -GACCCACTTCT*TTTCCTTGTC-3 21 base pairings; the control group is not clean 5 -GACCCACTTCATT*ATTTTCCTTGTC-3 25 base pairings; the f sig value is comparable with DNA7 group Note: Mismatched bases are underlined; T* alkyne-modified thymine. S23

25 S13. Supporting information references [1] S. Cheley, G. Braha, X. F. Lu, S. Conlan, H. Bayley, Protein Sci. 1999, 8, [2] S. Wen, T. Zeng, L. Liu, K. Zhao, Y. Zhao, X. Liu, H.-C. Wu, J. Am. Chem. Soc. 2011, 133, [3] D. Harries, D. C. Rau, V. A. Parsegian, J. Am. Chem. Soc. 2005, 127, [4] S. Moghaddam, C. Yang, M. Rekharsky, Y. H. Ko, K. Kim, Y. Inoue, M. K. Gilson, J. Am. Chem. Soc. 2011, 133, [5] M. Soskine, A. Biesemans, B. Moeyaert, S. Cheley, H. Bayley, G. Maglia, J. Am. Chem. Soc. 2013, 135, [6] J. Lagona, P. Mukhopadhyay, S. Chakrabarti, L. Isaacs, Angew. Chem. Int. Ed. 2005, 44, ; Angew. Chem. 2005, 117, [7] D. M. Bailey, A. Hennig, V. D. Uzunova, W. M. Nau, Chem. Eur. J. 2008, 14, [8] D. Rotem, L. Jayasinghe, M. Salichou, H. Bayley, J. Am. Chem. Soc. 2012, 134, S24