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 a, b, *, and Meni Wanunu a Department of Physics, Northeastern University, Boston, MA 5 b Department of Chemistry/Chemical Biology, Northeastern University, Boston, MA 5 c Anima Cell Metrology, Inc., Bernardsville, NJ 79 d Department of Chemistry, University of Pennsylvania, Philadelphia, PA 9 e Department of Physics, Arizona State University, Tempe, Arizona 858 f Biodesign Institute, Arizona State University, Tempe, Arizona 858 g Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 858 *Corresponding Author. E-mail: wanunu@neu.edu. Fax: (7) 7 9 S
Table of Contents Fig. SI: Scheme of voltage-induced trna deformation.. Fig. SI: Dwell time histograms for trna Arg at several voltages.. Fig. SI: Concatenated sample events for 5 trna..... Fig. SI: Contour plots of I/I o and dwell times for 5 trna, addition data set... Fig. SI5: Contour plots for trna Arg showing evolution over time... Table. SI: List of parameters used in SVM study of 5 trna... Fig. SI: Contour plots of trna Arg and trna Phe for mixture study...5 Table. SI: List of parameters used in SVM study of trna mixtures.......5 Table. SI: List of parameters used in SVM study of trna isoacceptors..... 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. SI: List of nanopores used and their sizes......7 S
Figure S. Cartoon of voltage-induced trna deformation/translocation through a 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 (t d ). mv 5 mv 5mV mv 5mV Log t d (µs) Figure S: Histograms for the log of dwell times for trna Arg 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 b. Location of peak values indicates most probable dwell times, where the standard deviation of the fit is propagated to find the error. S
Figure S: Traces showing concatenated events for each of the trna samples passing through a pore (~nm diameter, nm thickness) with an applied voltage of mv. Data was sampled at. MHz and shown after low-pass filtering at khz. Detected events are separated by, data points...8.... Phe N=9..8.... Arg N=7..8.... Thr N=9 8..8.... Tyr N=55 5..8.... 5 Ile N=85 5 Figure S: Contour plots depicting the fractional current blockage and dwell time of events for each of the trna samples passing through a pore (~nm diameter, 5nm thickness) with an applied voltage of 5mV. S
.7..5.... Arg Initial N=79 log t d (µs).7..5.... Arg Final N=9 5 log t d (µs) 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 (~9 minutes). Feature Name Description Unit Max Amplitude Greatest current blockade Spectrum Band 8 khz Log Spectrum Band 7 khz Log Spectrum Band 7 75 khz Log Spectrum Band 8 khz Dominant Frequency Strongest noise frequency khz Table S: Full list of features used in SVM analysis for discrimination of trna Phe, trna Arg, trna Tyr, trna Ile, and trna Tyr. S5
..8... Arg. 5..8... Phe. 5 5 Figure S: Comparison of trna Arg and trna Phe fractional current blockade and dwell time data used in mixture experiments. The two populations clearly display a very large degree of overlap. Feature Name Description Unit Peak Width Full width half amplitude ms Spectrum Band 9 khz Spectrum Band 8 khz Spectrum Band 8 khz OddEvenRatio Even wavenumbers divided by odd wavenumbers Log Spectrum Band 5 Log of power spectrum value between 5. - 5. khz Log Spectrum Band Log of power spectrum value between 5. - 5. khz Table S: Full list of features used in SVM analysis for discrimination of trna Phe and trna Arg mixtures. S
Feature Name Description Unit Spectrum Band 7 khz Spectrum Band khz Spectrum Band 9 khz Spectrum Band 5 9 khz Peak Width Full width at half amplitude ms Roughness Standard deviation current peak Table S: Full list of features used in SVM analysis for discrimination of trna Ile 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 : the power spectrum value at 8 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 Ile UAC trna molecules to Ile CAC trnas molecules. Data was collected at a sampling rate of.7mhz and shown after low pass filtering at khz. Experimental buffer contains mm KCl, mm tris, and mm EDTA. A voltage bias of 5mV is applied. Figure S9: All trna samples used in these experiments shown on a % denaturing PAGE gel. S8
Pore # Initial Size Experiments used Diameter ~. nm Voltage dependence Effective thickness ~5 nm (Fig., SI) Arg, Phe, Tyr, Thr, Diameter ~ nm Ile (Fig.,, SI, Effective thickness ~5 nm SI5) (Table, SI) 5 Diameter ~. nm Effective thickness ~9 nm Diameter ~. nm Effective thickness ~5 nm Diameter ~ nm Effective thickness ~ nm Arg & Phe mixture (Fig., SI) (Table SI) Val isoacceptors (Fig. 5, SI7) (Table SI) Arg, Phe, Tyr, Thr, Ile (Fig. SI) Table S: 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