SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFRMATIN doi: 1.138/nnano Supplementary Information Supplementary Methods Synthesis of aminocyclodextrin with a reactive linker arm, am 6 ampdp 1 βcd: To produce heptakis(6-deoxy-6-amino)-6-n-mono(2- pyridyl)dithiopropanoyl-β-cyclodextrin (am 6 ampdp 1 βcd), an ethanolic solution of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide, SPDP (25. mg, 8. µmol in 2.5 ml), was added dropwise to an aqueous solution of am 7 βcd (1 mg, 88.6 µmol in 2.5mL). The resulting solution was stirred at room temperature for 24 h. The mixture was then concentrated under reduced pressure at 6 C with a rotary evaporator to give a mixture of products: the unmodified cyclodextrin (am 7 βcd), the desired monosubstituted derivative (am 6 ampdp 1 βcd), and a small amount of polysubstituted cyclodextrins (i.e. am 5 ampdp 2 βcd etc.). Reverse-phase preparative HPLC was performed with a C 18 Gemini 5 µm 11A column (Phenomenex, 3 25 mm). The eluant was water containing.1% TFA and acetonitrile in a 9:1 ratio at min, with a linear gradient to 45:55 at 25 min, then a linear gradient to 9:1 at 3 min, then isocratic elution at 9:1 until 35 min. The flow rate was 22.5 ml min 1 at 25 C. Multiple injections of 2 µl (each containing ~1 mg of crude material) were performed. The eluate was analyzed by UV detection at 28 nm. The peak eluting at ~5 min was analysed by ESI-MS to confirm the absence of am 7 βcd starting material and then concentrated under reduced pressure nature nanotechnology 1

2 supplementary information doi: 1.138/nnano using a rotary evaporator (bath temperature 6 C) to give pure am 6 ampdp 1 βcd. Total yield 8.9 mg (4.7 %). The process was repeated by Chromatide Ltd. (Cheshire, UK) to produce larger quantities. The product was analysed on a Vydac C18 25 cm x.46 cm column in a gradient of acetonitrile in.1 % TFA at 1 ml/min with detection at 28 nm. The gradient was linear from 1-3 % acetonitrile for 15 min and 3-1 % acetonitrile in the next 5 min. The final purity was estimated to be 98% from the chromatogram (Supplementary Figure 3). The absence of unreacted am 7 βcd was confirmed by mass spectrometry. am 6 ampdp 1 -βcd.6tfa: 1 H-NMR (5 MHz, D 2, 25 C): δ 8.55 (d, 1H, J = 4.4 Hz), 8.1 (t, 1H, J = 7.8 Hz), 7.97 (d, 1H, J = 8.2 Hz), 7.55 (t, 1H, J = 6.2 Hz), (m, 7H), (m, 42H), (m, 2H), 2.79 (dt, 1H, J = 15.5, 6.3 Hz), 2.7 (dt, 1H, J = 15.5, 6.2 Hz); 13 C-NMR (1 MHz, D 2, 25 C): δ 173.7, (q, J = 35.3 Hz), 157.3, 146.5, 141.4, 122.9, 122.8, (q, J = Hz), [11.8, 11.6, 11.5], [82.6, 82.1, 82., 81.9, 81.5], [72.5, 72.2, 72.1, 72.], [71.8, 71.7, 71.6, 7.6], [67.8, 67.6], [4.3, 39.9, 39.8, 39.7, 39.4, 39.2], [34.3, 34.1]. HRMS (m/z): [M+H] + calcd for C 5 H 84 N 8 29 S 2, ; found , [M+Na] + calcd for C 5 H 84 N 8 29 S 2, ; found Production of cysteine mutants: All constructs presented in this work were assembled in the pt7-sc1 expression vector 1 and verified by DNA sequencing of the αhl inserts. Genes 2 nature nanotechnology

3 doi: 1.138/nnano supplementary information encoding the mutants were generated by PCR mutagenesis and ligation-free in vivo recombination as described elsewhere 2,3. Proteins were generated by coupled in vitro transcription and translation (IVTT) by using an E. coli T7-S3 extract system for circular DNA (Promega, no. L113). The complete amino acid mixture (1 mm) minus cysteine and the complete amino acid mixture (1 mm) minus methionine, supplied in the kit, were mixed in equal volumes to obtain the working amino acid solution required to generate high concentrations of the proteins. The amino acids (5. µl) were mixed with premix solution (2 µl), [ 35 S]Lmethionine (1 µl, MP Biomedicals, no. 511H, 1175 Ci/ mmol, 1 mci/ ml), rifampicin (1 µl,.8 mg/ml), plasmid DNA (8 µl, 4 ng/µl) and T7 S3 extract (15 µl) 4. Synthesis was carried out for 1.5 h at 37 C to produce 5 µl of radiolabeled IVTT protein monomer with an approximate concentration of 5 µg/ml. Heterooligomers for analysis by single-channel recording, WT- M113R/N139Q (1 µl) and WT-M113R/N139Q/L135C-D8 (25 µl), were generated after production of the subunits by IVTT. The negative charge of the D8 tail of the WT-M113R/N139Q/L135C-D8 protein was used to alter the electrophoretic mobility of the assembled pore allowing the separation of heteroheptamers 4. The IVTT mixes were first centrifuged at 25, g for 1 min to separate insoluble debris. The two supernatants were mixed together prior to the addition of rabbit red blood cell membranes (1 µl, 2.5 mg protein/ml). DTT was added to a final concentration of 2 mm and the mixture nature nanotechnology 3

4 supplementary information doi: 1.138/nnano was incubated for 1 h at 37 C. Afterwards, the mixture was centrifuged at 25, Xg for 1 min and the supernatant was discarded. The membrane pellet was washed by resuspension in 2 µl MBSA (1 mm MPS, 15 mm NaCl, ph 7.4, containing 1 mg/ml bovine serum albumin) and centrifugation, again at 25, Xg for 1 min. After discarding the supernatant, the membrane pellet was dissolved in 75 µl of 1X Laemmli sample buffer without heating, loaded into a single well of a 5% polyacrylamide gel aged for at least 2 days and containing.1 % SDS. The gel was electrophoresed for 18 h at 5 V with.1 mm sodium thioglycolate in the running buffer of the upper gel tank. The gel was then vacuum-dried onto a Whatman 3 MM filter paper at 5 C for about 3 h and then exposed to an X-ray film for 2 h (Supplementary Figure 4). The band containing WT-(M113R/N139Q) 6 (M113R/N139Q/L135C- D8) 1 was excised from the gel by using the autoradiogram as a template. The gel slice was rehydrated in 3 µl TE buffer (1 mm Tris.HCl, 1 mm EDTA, ph 8.) containing 2 mm DTT. After removing the filter paper, the gel piece was crushed using a sterile pestle. The protein was separated from gel debris by centrifugation through a.2 µm cellulose acetate spin filter (catalogue no , microfilterfuge tube, Rainin) at 25, Xg for 3 min. The filtrate was stored in aliquots at -8 C. The final protein concentration was estimated to be 1 µg/ml. Event Detection and Peak Fitting: Event histograms were constructed by using the following procedure: 1) Two adjacent windows of W T data points were passed through the raw data (2 khz sample rate, 1 khz Bessel filtered). 2). The T-statistic (a measure of 4 nature nanotechnology

5 doi: 1.138/nnano supplementary information the statistical difference between two populations) between the windows was calculated at each point. 3) Edges were identified by detecting peaks in the T- statistic with a minimum width of data points P T and exceeding a given threshold of T T. 4) The data between edges were averaged to determine the mean current and duration of the event. 5) Histograms of the mean event current were plotted. A reduction in pore current was assigned as nucleotide binding if the event occurred from the cyclodextrin current level and if the event was greater than N data points. Typical values for event detection to produce a residual current histogram were W T = 8, P T = 3, T T = 1, N = 8. Each peak in the event histogram was manually fitted with a Gaussian probability distribution by iterating mean, standard deviation and amplitude fits (for each nucleotide distribution) starting with user estimated parameters. Base confidence values were calculated using standard statistical techniques (error functions). Supplementary Results Comparison to previous data: Repeats of the previous work on nucleotide discrimination using a transient adapter produced similar results to the published data 5. Single channel recordings of the RL2-(M113R) 7 pore with the am 7 βcd adapter produced four overlapping peaks of residual ionic current between pa, corresponding to dgmp, dtmp, damp and dcmp (Supplementary Figure 5). Single nucleotide experiments confirmed that dgmp produced the largest current block, followed by dtmp, damp and dcmp. nature nanotechnology 5

6 supplementary information doi: 1.138/nnano For sequencing applications, a high proportion of the released nucleotides must be observed with sufficient data points to allow identification of the nucleotide with high confidence. Fast acquisition rates are therefore required to detect short nucleotide binding (~ 98% of all events are seen at 2 khz compared to ~ 67% at 1 khz). Accurate nucleotide discrimination can be achieved from the RL2-(M113R) 7 mutant when long events are considered (by filtering at 3 Hz as published). However, single channel recordings from the RL2-(M113R) 7 mutant at acquisition rates of 2 khz demonstrated that the resolution was not sufficient to identify a short nucleotide binding event with high enough confidence for sequencing. This led us to investigate the importance of various mutations within the protein in an attempt to improve the nucleotide discrimination. Importance of the protein background: As presented above, the RL2-(M113R) 7 mutant shows dnmp discrimination as previous reported. However, when a WT sequence was used the four nucleotides could not be resolved (Supplementary Figure 5). Nucleotide binding differed strongly between the two backgrounds with residual pore currents of pa and pa for the WT and RL2 backgrounds respectively. As reported elsewhere, the RL2 background contains five mutations with respect to the WT sequence 6. riginally, the four mutations in the transmembrane β barrel were chosen to minimize disruption to the pore. 6 nature nanotechnology

7 doi: 1.138/nnano supplementary information However, the data presented above indicate that one or more of the mutations affects base discrimination. We postulated that the responsible mutation is N139Q, which is the only mutation in the barrel of RL2 with the amino acid side chain orientated towards the hydrophilic lumen of the pore. To test this hypothesis, the (M113R/N139Q) 7 mutant was produced in the WT background and evaluated for base discrimination in the presence of the am 7 βcd adapter (Supplementary Figure 5). WT-(M113R/N139Q) 7 showed almost identical behaviour to RL2-(M113R) 7, confirming that the N139Q mutation is involved in base discrimination. Mechanistic evaluation: To understand the function of the M113R and N139Q mutations and the role of the cyclodextrin, a range of hemolysin pores were made, coupled to am 6 ampdp 1 βcd, and tested for nucleotide discrimination. WT- (M113R) 6 (M113R/L135C) 1, lacking the N139Q mutation, produced binding events but showed poor base discrimination. By contrast, WT- (N139Q) 6 (N139Q/L135C) 1, lacking the M113R mutation, produced very clean discrimination of the four nucleotides. (WT) 6 (L135C) 1, lacking both the M113R and the N139Q mutation, had a limited ability to discriminate nucleotides, but the frequency of events was very low (Supplementary Figure 6). These results show that the arginine side chains at position 113 of the β barrel 5 are not required for base discrimination when the location of the am 6 ampdp 1 βcd adapter is controlled. However, glutamine at the N139Q position improves base discrimination greatly. The reactive am 6 ampdp 1 βcd nature nanotechnology 7

8 supplementary information doi: 1.138/nnano is attached, via a disulfide bond, to a cysteine residue projecting into the lumen of the hemolysin β barrel. If the H groups on the secondary hydroxyl face of the am 6 ampdp 1 βcd were to stabilize the cyclodextrin through hydrogen bonding to glutamines at the 139 position 7, they would need to be in close proximity to the carbonyl groups of the amino acid side chains. When the adapter is covalently attached within the β barrel, we estimated that the vertical distance between the secondary hydroxyl face of the adapter and the thiol group on the linker to be 9.2 Å (Fig 2). For various cysteine mutants, the distance parallel to the axis of the β barrel between the C α of the cysteine residue and the C α of residue 139 was estimated using the wild-type crystal structure 8 (Fig 2): G119C (2.6 Å), N121C (2.9 Å), N123C (1.3 Å), T125C (13.5 Å), G133C (17. Å), L135C (9.1 Å) and G137C (7. Å). Therefore, molecular modelling predicts that the L135C mutation, which shows the best base discrimination, is also the most favourable to promote hydrogen-bond formation between the covalently attached cyclodextrin and the nanopore. The replacement of am 6 ampdp 1 βcd with the am 1 PDPβCD adapter produced a fluctuating baseline with a low frequency of nucleotide binding events, demonstrating that the primary amines on the adapter are required for base detection. Studies conducted over a range of ph values from 6. to 8.5 indicated that nucleotide detection efficiency drops off at more basic ph values (data not shown). It seems likely that partial deprotonation of the adapter occurs under these conditions. ne possibility is that the positively charged amino groups interact directly with the nucleotides, either through an electrostatic interaction with the phosphate group, or through π-cation 8 nature nanotechnology

9 doi: 1.138/nnano supplementary information interactions. Alternatively, the charges on the cyclodextrin molecule may result in a conformational change to the adapter, which allows more efficient nucleotide capture. Supplementary Figure Listing Supplementary Figure 1: Chemical structures of the am 7 βcd, am 6 ampdp 1 βcd and ampdp 1 βcd adapters used in this study. Supplementary Figure 2: Residual current histograms of dnmp binding events for the WT-(M113R/N139Q) 6 (M113R/N139Q/L135C) 1 -am 6 amdp 1 βcd pore under various physical conditions. Supplementary Figure 3: Chromatogram and column conditions for purification of the am 6 ampdp 1 βcd. Supplementary Figure 4: Autoradiogram of some of the proteins used in this study in a 5% polyacrylamide gel containing.1% SDS. A chain of eight aspartic acids (D8 tail) was present on the C-terminus of the subunit containing a single cysteine. The D8 tail affects the migration of the heptameric protein in the gel and heptamers containing different combinations of monomers are resolved by this method. The protein containing only one cysteine (x = 1) is extracted from the gel and used for single-channel recordings. nature nanotechnology 9

10 supplementary information doi: 1.138/nnano Supplementary Figure 5: Residual current histograms for mutants of αhl showing varying abilities to discriminate between nucleotides. The RL2- (M113R) 7 showed limited base discrimination at data high acquisition rates. The corresponding mutant in the wildtype background, WT-(M113R) 7 (lacking the N139Q) mutation showed very poor discrimination. When the N139Q mutation was added to the wild-type mutant, WT-(M113R/N139Q) 7 the resolution was comparable to the RL2 background. The WT- (M113R/N139Q) 6 (M113R/N139Q/L135C) 1 mutant with a covalently attached adapter is added to show the improvement to nucleotide discrimination over runs using a transient adapter. Experimental conditions: 8 mm KCl, 25 mm Tris.HCl, 1 mm MgCl 2, ph 7.5, at +16 mv. The cis solution contained in addition 1 µm dgmp, 1 µm dtmp, 1 µm damp and 1 µm dcmp. Runs with a transient adapter adapter contained 4 µm am 7 βcd in the trans chamber. All traces were recorded for 1 min at 2 khz with a 1 khz filter; a minimum of eight data points per event was used to construct the histograms. Supplementary Figure 6: Additional single-channel current recordings. A) Recordings showing dnmp binding events to a variety of mutant pores after reaction with am 6 ampdp 1 βcd. B) Residual current histograms from the pores used in A. C). Recordings of the WT- (M113R/N139Q) 6 (M113R/N139Q/L135C) 1 mutant after reaction with the ampdp 1 -βcd adapter showing a noisy baseline before dnmp addition. 1 nature nanotechnology

11 doi: 1.138/nnano supplementary information References 1 Cheley, S. et al., Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel. Protein Eng 1 (12), (1997). 2 Jones, D.H., in PCR mutagenesis and recombination in vivo PCR primer: a laboratory manual, edited by C. W. Dieffenbach & G. S. Dveksler (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995), pp Howorka, S. & Bayley, H., Improved protocol for high-throughput cysteine scanning mutagenesis. Biotechniques 25, (1998). Howorka, S., Cheley, S., & Bayley, H., Sequence-specific detection of individual DNA strands using engineered nanopores. Nat Biotechnol 19 (7), (21). Astier, Y., Braha,., & Bayley, H., Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. J Am Chem Soc 128 (5), (26). Cheley, S., Braha,., Lu, X., Conlan, S., & Bayley, H., A functional protein pore with a "retro" transmembrane domain. Protein Sci 8 (6), (1999). Wu, H.C., Astier, Y., Maglia, G., Mikhailova, E., & Bayley, H., Protein nanopores with covalently attached molecular adapters. J Am Chem Soc 129 (51), (27). Song, L. et al., Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274 (5294), (1996). nature nanotechnology 11

12 supplementary information doi: 1.138/nnano H2N NH2 H2N H H H H NH2 H H H H2N H H H NH2 H H H H H2N NH am 7 -βcd Supplementary Figure 1 - NNAN89996 H2N H H H H2N H H NH2 H H H H H H H NH S S N am 6 ampdp 1 -βcd H H NH2 NH2 H H H H H H H H H H H H H H H H NH S S N ampdp 1 -βcd H H H H 2 12 nature nanotechnology

13 doi: 1.138/nnano supplementary information HL-(M113R/N139Q) 6 (M113R/N139Q/L135C) 1 am 6 ampdp 1 -βcd 2/5 mm KCl, 25 mm Tris.HCl ph mv dgmp dtmp damp dcmp = 99.4 % = 9.3 % = 9.9 % = % HL-(M113R/N139Q) 6 (M113R/N139Q/L135C) 1 am 6 ampdp 1 -βcd 4/4 mm KCl, 25 mm Tris.HCl ph mv dgmp dtmp damp dcmp = 99.9 % = 97.9 % = 98. % = % HL-(M113R/N139Q) 6 (M113R/N139Q/L135C) 1 am 6 ampdp 1 -βcd 8/8 mm NaCl, 25 mm Tris.HCl ph mv dgmp dtmp damp dcmp = 99.9 % = 99.7 % = 99.8 % = % Supplementary Figure 2 - NNAN89996 nature nanotechnology 13

14 supplementary information doi: 1.138/nnano Time (min) Sum Area % Area Analysis Conditions Column: Vydac C18.46 cm x 25 cm Buffer: A = Water with.1 % TFA B = MeCN with.1 % TFA Gradient: 1-3 % B / 15 min & 3-1 % B / 5 min Flow rate: 1 cm 3 /min Temp: Room temp Wavelength: 28 nm Supplementary Figure 3 - NNAN nature nanotechnology

15 doi: 1.138/nnano supplementary information x = x = 1 x = 2 x = 3 x = % Polyacrylamide Gel Containing.1 % SDS WT-(M113R/N139Q) 7-x (M113R/N139Q/N121C-D8) x WT-(M113R/N139Q) 7-x (M113R/N139Q/N123C-D8) x WT-(M113R/N139Q) 7-x (M113R/N139Q/T125C-D8) x WT-(M113R/N139Q) 7-x (M113R/N139Q/G133C-D8) x WT-(M113R/N139Q) 7-x (M113R/N139Q/L135C-D8) x WT-(M113R/N139Q) 7-x (M113R/N139Q/G137C-D8) x Supplementary Figure 4 - NNAN89996 nature nanotechnology 15

16 supplementary information doi: 1.138/nnano RL2-(M113R) 7 (contains N139Q mutation) (transient adapter) Pore 1 Events: 35 Temp: 22.8 o C Dw: 1. ms Pore 2 Events: 3318 Temp: 23. o C Dw: 8.3 ms Pore 3 Events: 311 Temp: 23.3 o C Dw: 7.6 ms Supplementary Figure 5 - NNAN89996 WT-(M113R) 7 (lacks N139Q mutation) (transient adapter) Pore 1 Events: 3976 Temp: 22.4 o C Dw: 1.8 ms Pore 2 Events: 4586 Temp: 22.2 o C Dw: 12.2 ms Pore 3 Events: 5414 Temp: 22. o C Dw: 1.9 ms WT-(M113R/N139Q) 7 (transient adapter) Pore 1 Events: 251 Temp: 19.4 o C Dw: 1.8 ms Pore 2 Events: 2661 Temp: 21.2 o C Dw: 8.5 ms Pore 3 Events: 233 Temp: 21.7 o C Dw: 9.4 ms WT-(M113R/N139Q) 7 (M113R/N139Q/L135C) 1 (permanent adapter) Pore 1 Events: 9666 Temp: 22. o C Dw: 9.4 ms Pore 2 Events: 7987 Temp: 22.7 o C Dw: 8.1 ms Pore 3 Events 7935: Temp: 21.1 o C Dw: 9.9 ms 16 nature nanotechnology

17 doi: 1.138/nnano supplementary information a Time (s) Time (s) HL-(wt) 6 (L135C-D8) 1 HL-(N139Q) 6 (N139Q/L135C-D8) Time (s) Time (s) HL-(M113R) 6 (M113R/L135C-D8) 1 HL-(M113R/N139Q) 6 (M113R/N139Q/L135C-D8) 1 b Event Count Event Count HL-(wt) 6 (L135C-D8) HL-(N139Q) 6 (N139Q/L135C-D8) Event Count 6 4 Event Count HL-(M113R) 6 (M113R/L135C-D8) 1 HL-(M113R/N139Q) 6 (M113R/N139Q/L135C-D8) 1 c Supplementary Figure 6 - NNAN Time (s) HL-(M113R/N139Q) 6 (M113R/N139Q/L135C-D8) 1 ampdp1-βcd nature nanotechnology 17