Supporting Information
© Wiley-VCH 2008
69451 Weinheim, Germany
Supporting Information
Chemical Tags Facilitate the Sensing of Individual
DNA Strands with Nanopores
Nick Mitchell and Stefan Howorka *
[∗] Nick Mitchell and Dr. S. Howorka, Department of Chemistry, University College London,
London WC1H 0AJ, England (UK)
E-mail: [email protected]
Fax: +44 (0)20 7679 7463
1
Table of Contents
Experimental section
Page
1) Preparation of DNA strands carrying a single peptide tag
3
1.1. Peptide synthesis
3
1.2. Coupling of cysteine-terminated peptide to SPDP-activated DNA
4
2) Generation of POCs with two peptide tags
7
3) Single channel current recording
9
Results
4) Translocation events of chemically modified DNA
10
Discussion
5) Combined use of chemically modified DNA and solid-state nanopores
13
2
1) Preparation of DNA strands carrying a single peptide tag
Peptide-oligonucleotide conjugates were generated following Scheme 1. A DNA oligonucleotide
carrying an internal primary amino group was first reacted with the heterobifunctional cross-linker Nsuccinimidyl 3-(2-pyridyldithio)-propanoate (SPDP) leading to the formation of an amide bond. The thiolreactive pyridyldithio group was then coupled to a cysteine bearing peptide to form a disulfide bond
yielding the peptide-oligonucleotide conjugate.
5' -DNA
O
O
O
HN
O
O P O
O N
O
O
NH2
N
H
S
S
N
HN
O
O P O
O N
O
O
-
SPDP
O
O
O P O
O
O
5' -DNA
O
N O
Na-Phosphate pH 9.0
-
DNA-3'
O
N
H
H
N
S
S
N
O
O
O P O
O
DNA-3'
Tris pH 8.0
SH
O
5' -DNA
HN
O
O P O
O N
O
O
Peptide
O
N
H
H
N
S
S
O
Peptide
O
O P O
O
DNA-3'
Scheme 1. The generation of DNA oligonucleotides carrying a single peptide strand at an internal base position.
1.1. Peptide synthesis
Peptides were synthesized using standard Fmoc solid-phase peptide synthesis (SPPS) on a Syro
automated system using pre-loaded Wang resin and HBTU coupling chemistry. After completion of the
synthesis, peptides were deprotected and cleaved from the resin by incubation in TFA/TES/EDT/H2O
for 3 hours. The peptide solution was then collected, mixed with diethyl ether and allowed to precipitate
at -20°C. The suspension was centrifuged at 4000 rpm to a pellet and re-dissolved in diethyl ether. This
purification process was repeated three times after which the precipitate was dissolved in ddH2O,
frozen in liquid N2 and freeze-dried overnight. The peptides were analyzed and if necessary purified via
reversed phase HPLC using a Varian ProStar system with a Model 210 solvent delivery module and a
Model 320 UV detector. The preparative purification was performed using a Varian column (100 x 21.2
mm, 5 µm beads, flow rate of 10 mL/min) loaded with 100 µL aliquots of a 20 mg/mL solution of peptide
dissolved in 0.1% TFA containing water. The mobile phase was a linear gradient beginning with 95:5
water (0.1% TFA) / acetonitrile (0.1% TFA) to 80:20 water (0.1% TFA) / acetonitrile (0.1% TFA) over 10
min. The analysis of the chromatograms was conducted using Star Chromatography Workstation
software Version 1.9.3.2. The fractions containing the correct peak were pooled, the solvent removed
under reduced pressure to approximately 2 mL and the solution freeze-dried overnight. The resulting
white powder was analyzed via HPLC using a Gemini column (250 x 4.6 mm, 5 µm beads, flow rate of
1 mL/min) using the solvent system and gradient detailed above. ESI-MS analysis was performed on a
Waters Acquity Ultra Performance LC/MS machine. Figures 1A and 1C show the representative RP-
3
HPLC traces of the purified peptides with the sequence HOOC-His-His-His-His-His-His-Cys-NH2 and
HOOC-Gly-Tyr-Tyr-Tyr-Cys- NH2, respectively. Figures 1B and 1D display the ESI+ MS spectra for the
two peptides, respectively. The results of the HPLC and MS analysis for all peptides are summarized in
Table 1.
B
Abs. 214nm / A.U.
A
800
600
400
200
0
5
10
15
Retention time / min
D
Abs. 214 nm / A.U.
C
4000
3000
2000
1000
0
5
10
15
20
Retention tim / min
Figure 1. Analysis of peptides His6Cys1 and Gly1Tyr3Cys1. (A) RP-HPLC trace of His6Cys1. (B) ESI-MS spectrum of
His6Cys1. m/z expected 944.00, m/z found 944.36. (C) RP-HPLC trace of peptide Gly1Tyr3Cys1. (D) ESI-MS
spectrum of Gly1Tyr3Cys1. m/z expected 667.73, m/z found 668.18.
1.2. Coupling of cysteine-terminated peptide to SPDP-activated DNA
Synthetic DNA oligonucleotides carrying one amine-modified base analogue at an internal position
were first activated with SPDP and then reacted with one cysteine containing peptide (Scheme 1).
Three DNA oligonucleotides (IDT DNA, Coralville, IO) were used: 27-mers oligonucleotide O1 with the
sequence 5'-ACA TTC CTA AGT TCT GAA ACA TTA CAG-3', oligonucleotide O1-term with the same
sequence but an terminal amine C6 modifier rather than an internal modified base, and oligonucleotide
O2 with the sequence 5'-TCA ACT CGA TAC GTA CCT ATC AAT CTA-3'. The underlined T indicates
the thymine analogue carrying a primary amine (Scheme 1). For the activation, a 1 mM solution of
oligonucleotide (25 µL, 25 nmol) in 10 mM Tris, 1 mM EDTA, pH 8.0 was diluted with 0.1 M NaHCO3,
50 mM NaCl, pH 9.0 (65 µL). The mixture was combined with a 250 mM solution of SPDP in DMSO (10
µL, 2.5 µmol) leading to a white precipitate which immediately re-dissolved upon agitation. The reaction
was incubated at room temperature for 1 h. Unreacted SPDP was removed by size exclusion
chromatography using an AKTA purification system. A Sephadex column with a volume of 5 mL was
4
loaded with 100 µL of the reaction mixture at a flow rate of 5 mL/min. The mobile phase was buffer A
(20 mM Tris, 50 mM NaCl, pH 8.0), and the DNA containing fraction was collected using an isocratic
gradient.
Table 1. Chemical Characterization of Peptides and Peptide-Oligonucleotide Conjugates (POCs)
Oligonucleotide
Name
[a]
O1
Peptide
Length
[nt]
27
Sequence
POC
[c]
Mass [m/z]
AEC retention
time [min] /
[f]
purity [%]
Expected
Observed
n.a.
n.a.
n.a.
8380.6
[i]
9411.2
9410.8
24.1 / 96
9746.6
9759.03
25.4 / 75
[b]
n.a.
[e]
Mass [m/z]
HPLC
retention
time
[d]
[min]
O1
27
H6C1
944.00
O1-term.
27
G3H6C1
1253.00
944.36
627.82
[g]
9.7
15.0
[j]
Expected
Observed
8379.4
25.5 / 99
O1
27
H4C1
669.72
670.50
5.2
9136.1
9134.6
24.5 / 93
O1
27
H2C1
395.44
396.33
4.2
8862.6
8862.4
24.5 / 99
O1
27
G6C1
463.47
486.41
[h]
3.9
8930.8
8930.5
24.1 / 98
O1
27
G4C1
349.36
372.24
[h]
3.8
8816.2
8815.2
24.2 / 98
3.7
24.1 / 99
13.1
O1
27
G2C1
235.26
258.31
[h]
O1
27
G1R7C1
1271.52
636.55
[g]
O1
27
G1Y3C1
667.73
668.18
O2
27
G1Y3C1
667.73
O3
37
2xG1Y3C1
667.73
8702.3
8703.8
[k]
9738.6
9737.9
21.3 / 99
13.3
[l]
9134.6
9135.6
27.4 / 96
668.18
13.3
[l]
9149.6
9148.1
27.4 / 99
668.18
13.3
[l]
13105
13102
30.4 / 99
13098
13098
29.0 / 99.3
18160
18161
28.8 / 96.8
O5
37
2xG1Y3C1
667.73
668.18
13.3
[l]
O4
54
2xG1Y3C1
667.73
668.18
13.3
[l]
n.a.: not applicable.
[a]
Sequences of oligonucleotides: O1, 5'-ACA TTC CTA AGT TCT GAA ACA TTA CAG-3'; O2, 5'-TCA ACT CGA TAC GTA CCT
ATC AAT CTA-3'; O3, 5'-Phosph-TCA ACT CGA TAC GTA CCT ATC AAT CTA GTA CCT ATC A-3'. O5, 5’-Phosph-TCA ACT
CGA TAC GTA CCT ATC TAT CTA GTA CCT ATC A – 3’, O4, 5’ – ACA TTC CTA ACA TCA CTA ACT AAT CTT TCA ACT CGA
TAC GTA CCT ATC AAT CTA – 3’. The underlined T’s indicate amine-modified thymidines.
[b]
The peptide sequence is from the C to the N terminus.
[c]
Peptide masses were obtained in the ESI+ mode. Peptides were dissolved in HPLC grade water containing 0.1% TFA.
[d]
Unless otherwise stated, retention times are for HPLC analysis using C18 column (250 mm x 4.6 mm) and mobile phases 0.1%
TFA in water and 0.1% TFA in acetonitrile with a gradient of 5 % to 20 % acetonitrile/TFA over 10 min at a flow rate of 1 mL/min.
[e]
Oligonucleotide masses were obtained with MALDI–TOF using THAP as matrix and ammonium acetate as co-matrix.
[f]
Anion exchange chromatography (AEC) was performed using a Resource Q column (1 mL) and buffer B (20 mM Tris, pH 8.0)
and buffer C (buffer B plus 2 M NaCl) with a linear gradient from 0 to 40 % B over 45 min at a flow rate of 1 mL/min.
[g]
M2+ ion.
[h]
Peptide mass for the monosodium ion.
[i]
Gradient of 3 % to 10 % acetonitrile/TFA over 20 min.
[j]
Gradient of 2 % to 50 % acetonitrile/TFA over 20 min.
[k]
Gradient of 2 % to 60 % acetonitrile/TFA over 20 min.
[l]
Gradient of 2 % to 98 % acetonitrile/TFA over 20 min.
Cysteine-terminated peptide was covalently coupled to SPDP-activated DNA oligonucleotides.
Peptide (5 µmol) was dissolved in ddH2O containing 0.1 % TFA (100 µL, final concentration of 50 mM).
An aliquot (10 µL, 0.5 µmol) was combined with an aqueous solution of purified SPDP-activated DNA (2
mL, 25 nmol). In the case of R7C1-O1, 50% v/v of formamide was added to the buffer containing the
SPDP-activated oligonucleotide and the peptide to improve the solubility of the conjugate. The peptide
employed in the preparation of H6C1-O1-term had a maleimide group coupled to its N terminus to
enable the formation a thioether bridge between peptide and oligonucleotide. Therefore the disulfide
5
group on the SPDP-activated oligonucleotide was first reduced by incubation with 1000 eq. of
dithiothreitol overnight at 37 °C followed by purification via size exclusion chromatography. The
solutions for all peptide oligonucleotide mixtures were agitated on a shaker at room temperature for 2
hours followed by purification via anion exchange chromatography on an AKTA purifier system using a
Resource Q column (volume 1 mL). A linear gradient was employed beginning with buffer B (20 mM
Tris, pH 8.0) to 60:40 buffer B / buffer C (buffer B plus 2 M NaCl) over 45 min at a flow rate of 1 mL/min.
Product-containing fractions were desalted and concentrated to a volume of 200 µL using ultrafiltration
spin-columns (Amicon, 5 mL, 5KDa MW cut-off). The concentration of the purified conjugates was
determined using UV/vis spectroscopy (Cary Eclipse, Varian).
The purity of POCs was determined using analytical anion exchange chromatography. The method
for the chromatographic analysis was the same as described above for the conjugate preparation.
Representative traces of POCs His6Cys1-O1 and Gly1Tyr3Cys1-O2 are shown in Figure 2A and 2D,
respectively. Based on this analysis, the purity of the first POCs was > 96 % and > 99% for the second
POC. The results of the chromatographic analysis of all POCs are given in Table 1. POCs containing a
histidine peptide were also analyzed via immobilized metal affinity chromatography (IMAC) on an AKTA
system with a Ni-NTA containing His-Trap column (1 mL/min). The histidine-modified DNA was loaded
onto the column using ten column volumes of buffer D (0.1 M Na2HPO4, 50 mM NaCl, pH 8.0) and
eluted using a step gradient to buffer E (buffer D plus 1 M imidazole). A chromatogram for the IMAC
analysis of POC His6Cys1-O1 is shown Figure 2B.
For MALDI MS analysis, POCs were desalted via aspiration through C18 ZipTip pipette tips
(Millipore, cat # ZTC18S096) into a 0.1 M triethylammonium bicarbonate pH 8.0 buffer using a 50%
acetonitrile solution to elute the oligos. The desalted DNA solution was mixed with matrix solution
containing 2',4',6'-trihydroxyacetophenone (THAP) (2 µL, 50 mg/mL in methanol) and 0.5 µL of the comatrix ammonium acetate (0.1 M). The mixture was spotted onto the MALDI plate and the sample was
allowed to crystallize before being analyzed via MALDI MS using a Waters Micro MX machine. Figure
2C and 2E display the MALDI-MS spectra of His6Cys1-O1 and Gly1Tyr3Cys1-O2. The m/z values are
within 0.004 % and 0.016 % of the expected masses, respectively. Table 1 summarizes the MS
analysis for all POCs.
6
B
A
C
150
100
50
0
10
20
30
40
Retention time / min
4698.17
4676.10
300
0
(
4718.18
4701.69
600
%
200
Abs. 260 nm / A.U.
Abs. 260nm / A.U.
g
0
10
20
30
Retention time / min
6
4719.69
4734.70
9476.99
9379.95
9482.55
9347.76
9487.12
9492.83
9500.83
4664.98
2500
)
9410.77
9395.14
9451.33
9387.47
5000
7500
10000
12500
1500
E
D
Abs. 260nm / A.U.
150
100
50
0
10
20
30
40
Retention time / min
Figure 2. Chemical analysis of His6Cys1-O1 and Gly1Tyr3Cys1-O2. (A) Anion exchange chromatography trace of
His6Cys1-O1. (B) Immobilized metal affinity chromatography trace of His6Cys1-O1. (C) MALDI-MS spectrum of
His6Cys1-O1, m/z expected 9411.2, m/z found 9410.8. (D) Anion exchange chromatography trace of Gly1Tyr3Cys1O2. (E) MALDI-MS spectrum of Gly1Tyr3Cys1-O2, m/z expected 9149.6, m/z found 9148.1.
2) Generation of POCs with two peptide tags
Three peptide-oligonucleotide conjugates carrying two Y3 peptide tags each were prepared. The POCs
differed by the distance in nucleotides between the two peptide-tagged bases. The first two conjugates
Tyr3/Tyr3-O3 and Tyr3/Tyr3-O5 were 37-mer DNA oligonucleotides with a distance of 13 and 7 nt,
respectively, between the modified bases. The third POC Tyr3/Tyr3-O4 was a 54-mer and a separation
of 27 nt. For the preparation of the two 37-mers Tyr3/Tyr3-O3 and Tyr3/Tyr3-O5, DNA oligonucleotides
with the sequence 5'-Phos/TCA ACT CGA TAC GTA CCT ATC AAT CTA GTA CCT ATC A-3' and 5'Phos/TCA ACT CGA TAC GTA CCT ATC TAT CTA GTA CCT ATC A–3' were used, respectively. Two
Gly1Tyr3Cys1 tags were attached to the two amine-modified thymine analogues (underlined) using a
similar procedure used for the preparation of single-modified POCs. The anion exchange
chromatography trace and MALDI-MS spectrum of Tyr3/Tyr3-O3 are displayed in Fig. 3A and 3B,
respectively. The analysis data for Tyr3/Tyr3-O5 are summarized in Table 1.
7
A
Abs. 260 nm / A.U.
B
150
0
%
75
20
30
40
Retention time / min
5
6564.72
6558.58 6572.29
13102.35
6579.16 13085.31 13133.45
6553.61
13150.51
13066.29
1078.61
1079.66
5000
10000
15000
Figure 3. Analysis of Tyr3/Tyr3-O3. (A) Anion exchange chromatography trace, and (B) MALDI-MS spectrum,
expected m/z, 13105; found m/z 13102.
The double-modified peptide-oligonucleotide Tyr3/Tyr3-O4 was synthesized following a procedure
outlined in Scheme 2.
Scheme 2. Generation of POC Tyr3/Tyr3-O4 carrying two Gly1Tyr3Cys1 peptide strand. The procedure involves (1)
the modification of two separate oligonucleotides with SPDP and the two peptides, (2) the hybridization of the
Gly1Tyr3Cys1-O4A and Gly1Tyr3Cys1-O4B with a bridging 20-mer DNA oligonucleotide O4C and ligation with T4
Ligase, and (3) the isolation of the ligated POC Tyr3/Tyr3-O4 carrying two peptide tags with denaturing poly
acrylamide gel electrophoresis (PAGE).
The synthesis of Tyr3/Tyr3-O4 was performed with 27-mer oligonucleotides O4A of sequence 5'-ACA
TTC CTA ACA TCA CTA ACT AAT CTT-3' and O4B of sequence 5'-/5Phos/TCA ACT CGA TAC GTA
CCT ATC AAT CTA-3'. The two internal amine-modified thymine bases (underlined) were activated with
SPDP and coupled to Gly1Tyr3Cys1 as described above. The chemical analysis of resulting POCs
Gly1Tyr3Cys1-O4A and Gly1Tyr3Cys1-O4B via anion exchange chromatography and MALDI MS is
shown in Figure 4A to Figure 4D. To ligate the two POCs, a bridging oligonucleotide O4C was added.
O4C with the sequence 5'-A TCG AGT TGA AAG ATT AGT T-3' was complementary to the 3' end of
O4A and to the 5' end of O4B. Equimolar amounts of Gly1Tyr3Cys1-O4A, Gly1Tyr3Cys1-O4B, and O4C
(12.5 nmol) were diluted in 50 mM Tris, 10 mM MgCl2, 1 mM ATP, pH 7.5 (370 µL). The mixture was
heated in a PCR block to 95 ºC for 2 min and cooled to room temperature. The duplex was ligated by
adding T4 ligase (16 µL, 6400 units) followed by incubation at room temperature for 16 hours. The
resulting 54-mer POC Tyr3/Tyr3-O4 was separated from bridging O4C using denaturing 15% PAGE.
The gel electrophoretic analysis of Tyr3/Tyr3-O4, Gly1Tyr3Cys1-O4A, and O4C is shown in Figure 4E.
8
B
150
4517.77
100
2
3
9119.25
0
4
2500
D
5000
7500
10000
12500
15000
4581.00
120
4584.75
4586.33
80
4578.63
40
%
Abs. 260 nm / A.U.
1
9071.52
9088.44
9104.82
4489.51
4486.36
1326.94
50
C
4575.07
1013.47
4587.91
9150.66
9144.52 9160.72
4589.30
4593.65
9131.13
4595.43
9130.29
9171.34
9172.46
9189.52
9193.44
4572.90
0
10
20
30
40
Retention time /mins
4.
8981.02
8974.77
1050.00
E
8995.22 9054.20
4490.00 4514.32
10
20
30
40
Retention time /mins
Figure
9028.94
8996.47 9041.49
4507.82
%
Abs. 260 nm / A.U.
A
Analysis
of
Gly1Tyr3Cys1-O4A,
5
2000
4000
6000
Gly1Tyr3Cys1-O4B,
8000
10000
and
12000
14000
Tyr3/Tyr3-O4.
(A)
Anion
exchange
chromatography trace of Gly1Tyr3Cys1-O4A. Purity 96.2%. (B) MALDI-MS spectrum of Gly1Tyr3Cys1-O4A. m/z
expected 9029.6, m/z found 9028.9 (C) Anion exchange chromatography trace of Gly1Tyr3Cys1-O4B. Purity 99.3%.
(D) MALDI MS spectrum of Gly1Tyr3Cys1-O4B. m/z expected 9150.6, m/z found 9150.7. (E) Gel electrophoretic
analysis of the double modified POC Tyr3/Tyr3-O4 with a length of 54 nt (lane 1), 27-mer Gly1Tyr3Cys1-O4A (lane
2), and bridging oligonucleotide O4C with a length of 20 nt (lane 3).
3) Single channel current recording
Single-channel current recordings were performed by using a planar lipid bilayer apparatus as
described.[1] Briefly, a bilayer of 1,2- diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids) was
formed on an aperture (80 µm in diameter) in a Teflon septum (Goodfellow Corporation, Malvern, PA)
separating the cis and trans chambers of the apparatus. Each compartment contained 2 M KCl, 50 mM
Tris-HCl, pH 8.0 unless otherwise stated. Gel-purified heptameric αHL protein (final concentration 0.01–
0.1 ng/ml) was added to the cis compartment, and the electrolyte in the cis chamber was stirred until a
single channel inserted into the bilayer. Transmembrane currents were recorded at a holding potential
of +100 mV (with the cis side grounded) by using a patch–clamp amplifier (Axopatch 200B, Axon
Instruments, Union City, CA). For analysis, currents were low-pass filtered at 10 kHz or 30 kHz and
sampled at 50 kHz or 100 kHz, respectively, by computer with a Digidata 1200 A/D converter (Axon
Instruments), as described.[2]
9
4) Translocation events of chemically modified DNA
Table 2. Characteristics of type II translocation events of HxC1-O1 and GxC1-O1 [a]
Modified DNA
medium blockade
[b]
high blockade
[c]
τoff-m [ms]
Am [%]
[b]
[c]
τoff-h [ms]
Ah [%]
[d]
O1
n.o.
n.o.
n.o.
n.o.
H6C1-O1
56.6
1.34
97.4
1.96
H4C1-O1
53.5
1.27
96.4
1.26
H2C1-O1
57.6
1.25
94.5
0.87
54.9
1.39
92.3
0.82
G6C1-O1
G4C1-O1
55.1
1.28
92.1
0.67
G2C1-O1
56.2
1.48
94.0
0.65
[a] The recordings were conducted at 2 M KCl, 20 mM Tris, pH 8.0, filtered at 10 kHz and sampled at 50
kHz unless stated otherwise. The number of events analyzed for each type of DNA was between 1500
and 2000. [b] The relative amplitude was calculated using A = (IOC - IBC) / IOC, where IOC and IBC are the
conductance from the open and blocked channel, respectively. IOC and IBC were derived using all-point
histograms. [c] The average duration represents the mono-exponential fits of the dwell-time histograms.
[d] Filtered at 30 kHz and sampled at 100 kHz. n.o., not observed.
Table 3. Percentages of type I and type II events[a]
Modified DNA
Type I [%]
Type II [%]
Type I
with three steps between
92 and 99% level
O1
H6C1-O1
H4C1-O1
H2C1-O1
G6C1-O1
G4C1-O1
G2C1-O1
R7C1-O1
Y3C1-O1
Y3C1-O2
Y3/Y3-O3
Y3/Y3-O5
Y3/Y3-O4
100
n.o.
61
56
40
53
54
40
63
42
65
[b]
37
[b]
59
[b]
17
39
44
60
47
46
60
37
58
35
[c]
21
[c]
36
[c]
26
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
42
5
57
[a] Mid-amplitude events are excluded.
[b] Includes non-stepped events to the 99 % blockade level, as well as sloped and
single-step events between the 92 % and 99% level.
[c] Comprises events which start with the mid amplitude level and end at the 92 or
99% blockade level. n.o., not observed.
10
B
100
Number of events
Number of events
A
10
1
0 1 2 3 4 5 6 7 8 9 10 11
Dwell time / ms
90
80
70
60
50
40
30
20
10
0
0.80 0.85 0.90 0.95 1.00 1.05 1.10
Normalized amplitude / %
Figure 4. Dwell time histogram of H6C1-O1 type I events with single exponential fit (A) and amplitude histogram of
H4C1-O1 type I events (B). Dwell times were obtained by analyzing single channel current traces with Clampfit 9.0.
Event amplitudes were determined by subjecting individual blockades from single channel current traces to all-point
histogram analysis and determining the peak maxima by fitting to a Gaussian distribution. The magnitude of the
amplitude was normalized to the open channel using the formula (IOC - IBC) / IOC, where IOC and IBC are the
conductance levels from the open and blocked channel, respectively. The width of the amplitude of the distribution
in (B) is 2.1 % expressed as the standard deviation. The standard deviation was 0.5 % for R7C1-O1, and ranged
between 1.7 and 2.6 % for the remaining modified DNA strands.
Figure 5. Translocation events of O1, H6C1-O1, R7C1-O1, Y3C1-O1, Y3/Y3-O3, Y3/Y3-O4, and Y3/Y3-O5. Horizontal
scale bars represent a duration of 10 ms for O1, H6C1-O1, R7C1-O1, and 5 ms for Y3C1-O1, Y3/Y3-O3, Y3/Y3-O4,
and Y3/Y3-O5. The horizontal scale bars are aligned with the 0 pA level. Vertical bar, 100 pA.
O1
H6C1-O1
H6C1-O1
11
R7C1-O1
R7C1-O1
Y3C1-O1
Y3C1-O1
Y3/Y3-O3
Y3/Y3-O3
Y3/Y3-O4
Y3/Y3-O4
Y3/Y3-O5
Y3/Y3-O5
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5) Combined use of chemically modified DNA and solid-state nanopores
The general approach of using chemically modified bases is relevant for DNA sensing with solid-state
nanopores. Unlike lipid-bilayer embedded protein pores, inorganic pores exhibit very high mechanic
stability which makes them ideally suited for rugged electrical sensor devices. Despite progress in their
fabrication, solid-state nanopores cannot yet be engineered to the same atomic precision as the protein
pore αHL. This leaves many sensing approaches which are based on targeted chemical modification of
engineered cysteine residues inaccessible for inorganic pores. One approach to detect single-point
mutations with non-engineered pores uses the mismatch / perfect match-dependent formation of
duplexes.[3, 4] The perfect-matched wider double stranded duplexes give rise to long blockades which
can be distinguished from the shorter translocations events stemming from non-hybridized slimmer
single stranded DNA.[3, 4]
With regard to solid-state nanopores, this hybridization approach has two limitations which can be
addressed with the use of chemically modified DNA. The first limitation relates to the requirement that
the pore diameter has to be finely tuned. The pore should permit the passage of non-hybridized ssDNA
(0.8 nm diameter) but block duplexed DNA (2 nm). Reflecting this high demand on the fabrication
process, it has only been recently possible to generate an inorganic pore capable of discriminating
between ssDNA and short segments of duplexed DNA.[5] Due to inherent challenges within the
fabrication processes, it might not be possible to reach the critical dimensions for all solid-state
nanopores. The use of chemically modified DNA addresses this limitation by tuning the cross-sectional
DNA diameter to existing pore dimensions rather than matching the pore dimensions to the size of the
DNA strand. This is advantageous because it may be experimentally easier to attach bulky tags to
bases than to reduce the diameter of the solid-state pore below 2.0 nm. The second restraint of the
hybridization approach is that the current measurements have so far only been able to sense a single
base position per DNA strand. The sensing of two separate bases would require the binding of two
matching oligonucleotides and their sequential stripping off upon translocation through the pore. This
has so far not been demonstrated. By contrast, our approach on the use of chemical tags permits to
sense multiple bases in a DNA strand. It is noted that other approaches exist which use solid-state
nanopores for detecting single-point mutations. These include the use of specialized nucleic acid
binding proteins[6,
7]
or the method of discriminating translocating single and double stranded DNA
segments by the corresponding different extents of current blockade.[8]
The concept of slowing down DNA via chemical tags can be applied to various other nanopore
approaches. For example, tags can be used for the fluorescence-based sequencing of DNA-derived
design polymers where the spacing between individual nucleotides has been increased.[9] This potential
application has been suggested in a recent review on single-molecule sequencing.[10] In extension of
LingVitae’s concept of enzymatically placing 4-40 nt long spacers between bases of a DNA strand, it
can be envisioned to chemically modify the four bases with different tags in order to discriminate the
bases based on their current signatures. This possible development would require technical
13
improvements in the tags. In particular, one aim would be to achieve four different current levels for the
four bases, e.g. by reducing the linker-induced steric flexibility between the tag and the base.
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J. Mathe, H. Visram, V. Viasnoff, Y. Rabin, A. Meller, Biophys. J. 2004, 87, 3205.
J. Nakane, M. Wiggin, A. Marziali, Biophys. J. 2004, 87, 615.
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