Download Supporting Information Electrophoretic Deformation of Individual Transfer RNA Molecules Reveals Their Identity

Supporting Information
Electrophoretic Deformation of Individual Transfer
RNA Molecules Reveals Their Identity
Robert Y. Henley a, Brian Alan Ashcroft f, Ian Farrell c, Barry S. Cooperman d, Stuart M. Lindsay
e, f, g
, and Meni Wanunu a, b, *
a
Department of Physics, Northeastern University, Boston, MA 02115
b
Department of Chemistry/Chemical Biology, Northeastern University, Boston, MA 02115
c
Anima Cell Metrology, Inc., Bernardsville, NJ 07924
d
Department
of
Chemistry,
University
of
Pennsylvania,
Philadelphia,
PA
19104
e
Department of Physics, Arizona State University, Tempe, Arizona 85281
f
Biodesign Institute, Arizona State University, Tempe, Arizona 85281
g
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85281
*Corresponding Author. E-mail: [email protected]. Fax: (617) 373 2943
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Table of Contents
Fig. SI1: Scheme of voltage-induced tRNA deformation……………….…….…………………2
Fig. SI2: Dwell time histograms for tRNAArg at several voltages…..……………………………2
Fig. SI3: Concatenated sample events for 5 tRNA……….…………..……..……………………3
Fig. SI4: Contour plots of ∆I/Io and dwell times for 5 tRNA, addition data set….........…………3
Fig. SI5: Contour plots for tRNAArg showing evolution over time…………….....………………4
Table. SI1: List of parameters used in SVM study of 5 tRNA…………………………………...4
Fig. SI6: Contour plots of tRNAArg and tRNAPhe for mixture
study................................................5
Table. SI2: List of parameters used in SVM study of tRNA mixtures………………..…..……...5
Table. SI3: List of parameters used in SVM study of tRNA isoacceptors……………..……..….6
Fig. SI7: Time evolution of various SVM parameters from tRNA isoacceptors………....………7
Fig. SI8: Raw current traces of isoacceptor mixtures……………………………………..………7
Fig. SI9: PAGE gel of 5 tRNA samples used……………………………………………..………7
Table. SI3: List of nanopores used and their sizes……………..………………………….…..….7
S2
Figure S1. Cartoon of voltage-induced tRNA deformation/translocation through a 3 nm diameter
nanopore. a) Trans-pore voltage induces a pore-localized electric field that drives a tRNA molecule
into the pore constriction; b) as the elbow region reaches the pore, electrophoresis through the
constriction induces tRNA deformation from its native conformation; c) Electro-deformed tRNA
translocates through the pore. Sample experimental pulse is inset to c), with markers indicating the
mean current blockade (∆Ι) and dwell time (td).
Counts
300
400mV
200
100
0
0
2
4
6
Counts
300mV
100
50
0
Counts
0
2
4
6
250mV
60
40
20
0
0
2
4
6
Counts
200mV
40
20
0
0
2
4
6
Counts
150mV
40
20
0
0
2
4
Log td (µs)
6
Arg
Figure S2: Histograms for the log of dwell times for tRNA molecules at different voltages fit to lognormal distributions. This data and the fits we’re used for the voltage vs. dwell time plot in Figure 2b.
Location of peak values indicates most probable dwell times, where the standard deviation of the fit is
propagated to find the error.
S3
0.8
0.6
0.4
0.2
0
0.0
0
1
10
2
20
30
3
4
1.0
Arg
N=7121
0.8
0.6
0.4
0.2
0
0.0
0
10
1
log td(µs)
Fractional Current Blockage
Fractional Current Blockage
Phe
N=961
2
20
30
3
4
1.0
Thr
N=2966
0.8
0.6
0.4
0.2
0
0.0
0
1
log td(µs)
1.0
Tyr
N=10515
0.8
0.6
0.4
0.2
0 100 200 300 400 500
0.0
0
1
2
3
log td(µs)
4
Fractional Current Blockage
1.0
Fractional Current Blockage
Fractional Current Blockage
Figure S3: Traces showing concatenated events for each of the tRNA samples passing
through a pore (~3nm diameter, 10nm thickness) with an applied voltage of 300mV. Data
was sampled at 4.166 MHz and shown after low-pass filtering at 300kHz. Detected events
are separated by 10,000 data points.
20
2
40
60
3
80 100
4
log td(µs)
1.0
Ile
N=3856
0.8
0.6
0.4
0.2
0
0.0
0
1
50
2
100 150 200
3
4
log td(µs)
Figure S4: Contour plots depicting the fractional current blockage and dwell time of events for each of
the tRNA samples passing through a pore (~3nm diameter, 5nm thickness) with an applied voltage of
500mV.
S4
Fractional Current Blockage
0.7
Fractional Current Blockage
0.7
0.6
Arg Initial
N=729
0.5
0.4
0.3
0.2
0
0.1
0
0.6
10
20
2
4
log td(µs)
30
6
Arg Final
N=924
0.5
0.4
0.3
0.2
0 10 20 30 40 50
0.1
0
2
4
log td(µs)
6
Figure S5: Contour plots depicting the fractional current blockage and dwell time of events for two
different runs of the Arg tRNA on the same pore with the same conditions. Taken at the beginning and
end of an experiment to show minimal pore expansion over the course of an experiment (~90
minutes).
Feature Name
Max Amplitude
Spectrum Band 4
Log Spectrum Band
13
Log Spectrum Band
27
Log Spectrum Band
28
Dominant Frequency
Description
Greatest current blockade
Power spectrum value at
833 kHz
Power spectrum value at
47 kHz
Power spectrum value at
275 kHz
Power spectrum value at
301 kHz
Strongest noise frequency
Unit
nA
nA
Unitless
Unitless
Unitless
kHz
Phe
Table S1: Full list of features used in SVM analysis for discrimination of tRNA
Ile
Tyr
tRNA , and tRNA .
Arg
, tRNA
Tyr
, tRNA ,
S5
Fractional Current Blockage
1.0
Arg
0.8
0.6
0.4
0.2
0
0.0
0
1
2
10
20
30
40
3
4
log td(µs)
5
6
Fractional Current Blockage
1.0
Phe
0.8
0.6
0.4
0.2
0
0.0
0
1
2
Arg
10
20
30
3
4
log td(µs)
40
50
5
60
6
Phe
Figure S6: Comparison of tRNA and tRNA
fractional current blockade
and dwell time data used in mixture experiments. The two populations clearly
display a very large degree of overlap.
Feature Name
Peak Width
Spectrum Band 6
Spectrum Band 8
Spectrum Band 10
OddEvenRatio
Log Spectrum Band 5
Log Spectrum Band 6
Description
Full width half amplitude
Power spectrum value at 1249
kHz
Power spectrum value at 1666
kHz
Power spectrum value at 2082
kHz
Even wavenumbers divided
by odd wavenumbers
Log of power spectrum value
between 5.1 - 5.12 kHz
Log of power spectrum value
between 5.12 - 5.16 kHz
Unit
ms
nA
nA
nA
Unitless
Unitless
Unitless
Phe
Table S2: Full list of features used in SVM analysis for discrimination of tRNA
Arg
and tRNA
mixtures.
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Feature Name
Spectrum Band 1
Spectrum Band 3
Spectrum Band 4
Spectrum Band 5
Peak Width
Roughness
Description
Power spectrum value at
17 kHz
Power spectrum value at
33 kHz
Power spectrum value at
39 kHz
Power spectrum value at
49 kHz
Full width at half
amplitude
Standard deviation current
peak
Unit
nA
nA
nA
nA
ms
nA
Ile
Table S3: Full list of features used in SVM analysis for discrimination of tRNA isoacceptors.
Figure S7: Time evolution of several SVM parameters during tRNA isoacceptor
experiment. a) Peak width: the full width at half max of a translocation spike. b)
Spectrum band 2: the power spectrum value at 18 kHz, measured during
translocation. c) Average amplitude: average current blocked during translocation.
S7
Figure S8: Raw current traces are shown from each of the mixture samples tested. Each
mixture is labeled to show the ratio of IleUAC tRNA molecules to IleCAC tRNAs molecules.
Data was collected at a sampling rate of 4.17MHz and shown after low pass filtering at
200kHz. Experimental buffer contains 400mM KCl, 10mM tris, and 1mM EDTA. A voltage
bias of 500mV is applied.
Figure S9: All tRNA samples used in these experiments shown on a 12%
denaturing PAGE gel.
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Pore #
1
Initial Size
Diameter ~ 3.2 nm
Effective thickness ~5 nm
2
Diameter ~ 3 nm
Effective thickness ~5 nm
3
Diameter ~ 3.2 nm
Effective thickness ~9 nm
4
Diameter ~ 3.2 nm
Effective thickness ~5 nm
5
Diameter ~ 3 nm
Effective thickness ~10 nm
Experiments used
Voltage dependence
(Fig. 1, SI2)
Arg, Phe, Tyr, Thr,
Ile (Fig. 2, 3, SI3,
SI5) (Table 1, SI1)
Arg & Phe mixture
(Fig. 4, SI6) (Table
SI2)
Val isoacceptors
(Fig. 5, SI7) (Table
SI3)
Arg, Phe, Tyr, Thr,
Ile (Fig. SI4)
Table S4: Full list of nanopores used, their initial sizes, and the experiments they were used for. Pore
sizes were estimated as described in the main text.
S9