4 Souvik Modi, Swetha M.G, Debanjan Goswami, Gagan D · PDF file48 addition of 3 μL of 0.5...

nature nanotechnology | www.nature.com/naturenanotechnology 1

supplementary informationdoi: 10.1038/nnano.2009.83

NNANO-08101037 1

Supplementary Information for the paper 1

A DNA nanomachine that maps spatial and temporal 2

pH changes inside living cells 3

Souvik Modi, Swetha M.G, Debanjan Goswami, Gagan D Gupta, Satyajit Mayor, 4

Yamuna Krishnan* 5

Methods. 6

Materials. HPLC purified DNA oligonucleotides were used without further 7

purification whereas fluorescently modified oligonucleotides were ethanol 8

precipitated prior to further use. Nigericin, biotin labelled human holo-Transferrin and 9

Streptavidin were obtained from Sigma. FITC labelled dextran, Lysotracker™ red, 10

Mitotracker® Green were obtained from Molecular probes, Invitrogen. All other 11

reagents were purchased from Sigma-Aldrich unless otherwise specified. 12

Sample Preparation. Stock DNA solutions were prepared by dissolving lyophilized 13

DNA in Milli-Q water and stored at -20oC until further use. 5 μM of O1, O2 and O3 14

were mixed in equimolar ratios in 20 mM potassium phosphate buffer of desired pH 15

containing 100 mM KCl. The resultant solution was heated to 90oC for 5 minutes, 16

cooled to the room temperature over 3 hr at 5oC/15 min and equilibrated at 4oC 17

overnight. Prior to experiment, the solution was diluted to 80 nM in appropriate buffer 18

containing 100 mM KCl unless otherwise mentioned. 19

Circular Dichroism spectroscopy. All CD scans were performed on a JASCO J-815 20

spectrophotometer equipped with a temperature controller. A 5 μM stock of the I-21

switch was annealed as described previously. Samples at pH 5 and 7.3 were prepared 22

at 20 mM potassium phosphate in 100 mM KCl as stated earlier. Samples were then 23

© 2009 Macmillan Publishers Limited. All rights reserved.

2 nature nanotechnology | www.nature.com/naturenanotechnology

supplementary information doi: 10.1038/nnano.2009.83

NNANO-08101037 2

diluted to 1 μM in the appropriate buffer and equilibrated for an hour and are 24

presented as an average of 5 successive scans. 25

Steady state fluorescence measurements. All fluorescence spectra were measured 26

on a Jasco-815 CD spectrometer with an inbuilt fluorimeter. Fluorescently labelled I-27

switch containing O1 labelled with Alexa 488 (O1-488) and O2 labelled with Alexa 28

647 (O2-647) were prepared as stock solution of 5 μM stock in 20 mM pH 5.5 29

potassium phosphate, 100 mM KCl. Samples were diluted to 80 nM in appropriate 30

buffer for all fluorescence experiments. The samples were excited at 488 nm and 31

emission collected between 505 nm-750 nm. FRET efficiencies were calculated using 32

the formula, E = 1 – IDA/ID = 1/[1+(R/Ro)6], where ID is the intensity of the donor in 33

the absence of the acceptor, IDA is the intensity of the donor in the presence of the 34

acceptor, Ro is the Förster’s distance and R is the interfluorophore distance. Ro for 35

Alexa 488 and Alexa 647 was taken as 50 Å. 36

FRET pair used in the study: We have used three different FRET pairs in this study. 37

Alexa 546-Alexa 647 was used to characterize the closing and opening of I-switch by 38

pH cycling as well as to confirm the conformation of the closed state. Bodipy TMR-39

Alexa 647 was used in the following cases only (i) a distinct FRET pair to confirm 40

that the pH profile of the I-switch is not due to dye sensitivity, but due to i-motif 41

formation (ii) colocalization studies of I-switch with FITC dextran, (iii) colocalization 42

studies of I-switch in Rab 5-GFP expressing hemocytes and (iv) to prove integrity of 43

the I-switch scaffold under the conditions used. For all other studies, Alexa 488-Alexa 44

647 labeled I-switch was used. 45

pH cycling. pH cycling experiments were performed on a Fluorolog-Spex 46

spectrophotometer. I-switch (5 nM) was cycled between pH 5 to pH by the alternate 47

addition of 3 μL of 0.5 N HCl and 4 μL of 0.5 N KOH respectively. 48




NNANO-08101037 2

diluted to 1 μM in the appropriate buffer and equilibrated for an hour and are 24

presented as an average of 5 successive scans. 25

Steady state fluorescence measurements. All fluorescence spectra were measured 26

on a Jasco-815 CD spectrometer with an inbuilt fluorimeter. Fluorescently labelled I-27

switch containing O1 labelled with Alexa 488 (O1-488) and O2 labelled with Alexa 28

647 (O2-647) were prepared as stock solution of 5 μM stock in 20 mM pH 5.5 29

potassium phosphate, 100 mM KCl. Samples were diluted to 80 nM in appropriate 30

buffer for all fluorescence experiments. The samples were excited at 488 nm and 31

emission collected between 505 nm-750 nm. FRET efficiencies were calculated using 32

the formula, E = 1 – IDA/ID = 1/[1+(R/Ro)6], where ID is the intensity of the donor in 33

the absence of the acceptor, IDA is the intensity of the donor in the presence of the 34

acceptor, Ro is the Förster’s distance and R is the interfluorophore distance. Ro for 35

Alexa 488 and Alexa 647 was taken as 50 Å. 36

FRET pair used in the study: We have used three different FRET pairs in this study. 37

Alexa 546-Alexa 647 was used to characterize the closing and opening of I-switch by 38

pH cycling as well as to confirm the conformation of the closed state. Bodipy TMR-39

Alexa 647 was used in the following cases only (i) a distinct FRET pair to confirm 40

that the pH profile of the I-switch is not due to dye sensitivity, but due to i-motif 41

formation (ii) colocalization studies of I-switch with FITC dextran, (iii) colocalization 42

studies of I-switch in Rab 5-GFP expressing hemocytes and (iv) to prove integrity of 43

the I-switch scaffold under the conditions used. For all other studies, Alexa 488-Alexa 44

647 labeled I-switch was used. 45

pH cycling. pH cycling experiments were performed on a Fluorolog-Spex 46

spectrophotometer. I-switch (5 nM) was cycled between pH 5 to pH by the alternate 47

addition of 3 μL of 0.5 N HCl and 4 μL of 0.5 N KOH respectively. 48

NNANO-08101037 3

Time resolved Fluorescence measurement. Time resolved experiments were 49

measured in Zeiss LSM 5 Meta multiphoton set up. Briefly, fluorophores excited by 50

multiphoton excitation were built on a Zeiss LSM 510 Meta microscope (Carl Zeiss, 51

http://www.zeiss.com) with 63× 1.4 numerical aperture (NA) objective coupled to the 52

femtosecond-pulsed Tsunami Titanium:Sapphire tunable pulsed laser. Fluorescence 53

was collected in Hamamatsu R3809U multi-channel plate photomultiplier tubes 54

(PMTs; Hamamatsu Photonics, http://www.hamamatsu.com) while TCSPC was 55

accomplished using a Becker & Hickl 830 card (Becker and Hickl, 56

http://www.becker-hickl). For multiphoton excitation of Alexa 488, 720 nm excitation 57

wavelength was used. The repetition rate of the pulsed laser is 80.09 MHz (12 ns). 58

The instrument response function (IRF) was calculated from 10–16-nm gold particles 59

dried on a coverslip as a second harmonic generator; full width at half maximum 60

(FWHM) of IRF is approximately 60 ps. Experimentally measured fluorescence 61

decay is a convolution of the IRF with the intensity decay function. Average lifetimes 62

at different pH were obtained from the intensity decay data were fit to the appropriate 63

equations by an iterative reconvolution procedure using a Levenberg-Marquardt 64

minimization algorithm34. 65

Cell culture and labelling. Hemocytes were obtained from wandering third instar 66

Drosophila larvae as described previously. Briefly, larvae were surface sterilized and 67

then punctured to release the hemolymph into complete insect medium that is 68

composed of Schneider’s insect medium supplemented with 10% non-heat inactivated 69

FBS, 1 �g/ml bovine pancreatic insulin, 150 �g/ml penicillin, 250 �g/ml 70

streptomycin, 750 �g/ml glutamine. The medium is then plated onto 35 mm coverslip 71

dishes. Labeling of cells with fluorophores was performed 1.5 hours after dissection. 72

Cells were washed with Medium 1 prior to labeling. For pH measurement 73

experiments, cells were imaged live, after chasing the probes for the stated timepoints. 74




NNANO-08101037 4

The pH standard curve was generated in cells by briefly fixing cells (for 1minute) 75

with 2.5% paraformaldehyde (PF) and then adding the ionophore, nigericin along 76

with medium 1 buffered to appropriate pH (ranging from 5-7). For colocalization 77

experiments cells were fixed with 2.5% Pf for 20 minutes. For colocalization of Rab5 78

with the sensor, transgenic flies expressing Rab5GFP were crossed to flies expressing 79

Heme-Gal4 and GFP positive progeny were used for hemocyte cell culture. Stable 80

line of Drosophila SR+ cells expressing the human Transferrin receptor (Gupta et al, 81

unpublished) were used for labeling experiments. Cells were labeled on ice with the 82

switch conjugated to biotinylated Transferrin (Sigma), and then pulsed at room 83

temperature for 15 minutes. Cells were then moved to ice and the treated with low pH 84

ascorbate buffer to remove the remaining receptor-bound ligands. The Transferrin 85

conjugated with the switch was imaged after fixation with 2.5% formaldehyde. 86

Fluorescence microscopy and image analysis. All the wide-field images were 87

collected using a Nikon inverted microscope equipped with 60×, 1.4 NA objectives, a 88

mercury arc illuminator (Nikon, Japan), and a cooled CCD camera (Andor, USA) 89

controlled by Metamorph software (Universal Imaging, PA). Three sets of images 90

were taken corresponding to (I) image at donor emission wavelength upon donor 91

excitation (donor image), (II) image at acceptor emission wavelength (acceptor 92

FRET) upon donor excitation and and (III) image at acceptor emission wavelength 93

(Acceptor image) acceptor excitation. Confocal imaging was carried out on an 94

Olympus Fluoview 1000 confocal microscope (Olympus, Japan) using an Argon ion 95

laser for 488 nm excitation and He-Ne laser for 543 excitation with a set dichroics, 96

excitation, and emission filters suitable for each fluorophore. Cross talk and bleed-97

through were measured with donor only and acceptor only species and found to be 98

negligible for Alexa 488-647 pair. Autofluorescence was measured on unlabelled 99

cells. All the images were then background subtracted taking mean intensity over a 100

large cell free area. Donor and acceptor images were colocalized and endosomes 101




NNANO-08101037 4

The pH standard curve was generated in cells by briefly fixing cells (for 1minute) 75

with 2.5% paraformaldehyde (PF) and then adding the ionophore, nigericin along 76

with medium 1 buffered to appropriate pH (ranging from 5-7). For colocalization 77

experiments cells were fixed with 2.5% Pf for 20 minutes. For colocalization of Rab5 78

with the sensor, transgenic flies expressing Rab5GFP were crossed to flies expressing 79

Heme-Gal4 and GFP positive progeny were used for hemocyte cell culture. Stable 80

line of Drosophila SR+ cells expressing the human Transferrin receptor (Gupta et al, 81

unpublished) were used for labeling experiments. Cells were labeled on ice with the 82

switch conjugated to biotinylated Transferrin (Sigma), and then pulsed at room 83

temperature for 15 minutes. Cells were then moved to ice and the treated with low pH 84

ascorbate buffer to remove the remaining receptor-bound ligands. The Transferrin 85

conjugated with the switch was imaged after fixation with 2.5% formaldehyde. 86

Fluorescence microscopy and image analysis. All the wide-field images were 87

collected using a Nikon inverted microscope equipped with 60×, 1.4 NA objectives, a 88

mercury arc illuminator (Nikon, Japan), and a cooled CCD camera (Andor, USA) 89

controlled by Metamorph software (Universal Imaging, PA). Three sets of images 90

were taken corresponding to (I) image at donor emission wavelength upon donor 91

excitation (donor image), (II) image at acceptor emission wavelength (acceptor 92

FRET) upon donor excitation and and (III) image at acceptor emission wavelength 93

(Acceptor image) acceptor excitation. Confocal imaging was carried out on an 94

Olympus Fluoview 1000 confocal microscope (Olympus, Japan) using an Argon ion 95

laser for 488 nm excitation and He-Ne laser for 543 excitation with a set dichroics, 96

excitation, and emission filters suitable for each fluorophore. Cross talk and bleed-97

through were measured with donor only and acceptor only species and found to be 98

negligible for Alexa 488-647 pair. Autofluorescence was measured on unlabelled 99

cells. All the images were then background subtracted taking mean intensity over a 100

large cell free area. Donor and acceptor images were colocalized and endosomes 101

NNANO-08101037 5

showing good colocalization were analysed using ImageJ and total intensity as well as 102

mean intensity in each endosome was measured in donor and acceptor channels. A 103

ratio of donor to acceptor intensities (D/A) was obtained from. Five independent 104

measurements were presented as the mean± standard error (s.e.m). 105

Ratiometric pH measurements. The in vitro pH calibration profile was obtained on 106

A JASCO-815 CD spectrometer. I-switch at 80 nM was prepared in buffers of 107

different pH values from 4 to 7.3. Measurements from two independent samples were 108

taken at given pH and the mean donor intensity (D) at 520 nm and mean acceptor 109

intensity (A) at 665 nm were recorded. D/A values at each pH were plotted as a 110

function of pH to give the in vitro calibration curve. Error bars were representative of 111

the standard error between five independent measurements. For the intracellular pH 112

calibration curve, cells were pulsed, chased for 5 min, washed, incubated with 10 μM 113

Nigericin in buffers at a given pH and then mildly fixed. Donor and acceptor FRET 114

images were acquired as described earlier from which D/A ratios were obtained. The 115

mean D/A of individual endosomes with associated s.e.m at each pH were plotted as a 116

function of pH for the intracellular pH calibration curve. Spatiotemporal pH changes 117

were followed by comparing the spread in D/A values of ~ 80 endosomes for 5 118

independent experiments at the specified chase time to the calibration curve. 119

Time dependent pH measurement data was generated by imaging different portions 120

on a single coverslip over 2 h with a time interval of 5 minutes each, such that each 121

time frame has five images covering five different places in the coverslip. From those 122

five fields of view, 40 endosomes at each time point were quantified and mean D/A 123

(for two independent experiment) ± s.e.m was calculated and plotted over time. 124

Protein Conjugation of I-switch. For conjugation to Transferrin, I-switch 125

incorporated with a biotin label was used.O1-488 and O2-647 was hybridized with 5’-126

Biotinylated O3 in pH 5.5 phosphate buffer. 5 μL of 5 μM biotinylated switch (IB) 127




NNANO-08101037 6

was diluted 2 fold with PBS pH 7.4 and to it 5 μL of Streptavidin (1 mg/ml) was 128

added slowly and incubated at room temperature for 1 hr. Conjugation (IB-SA) was 129

confirmed by agarose gel electrophoresis (3%). This conjugate (IB-SA) was further 130

conjugated with biotinylated holo-Transferrin (TfB) similarly in a 1:2 ratio. Excess 131

Biocytin (1 nanomole) was added after 1 h to the solution to prevent aggregation. 132

Formation of Transferrin conjugated I-switch (IB-SA-TfB) was confirmed by gel 133

electrophoresis. The presence of Transferrin with the sensor was confirmed by 134

colocalization experiments and by competing out the sensor with Alexa-568 labeled 135

as well as unlabeled Transferrin. 136

137

138




NNANO-08101037 6

was diluted 2 fold with PBS pH 7.4 and to it 5 μL of Streptavidin (1 mg/ml) was 128

added slowly and incubated at room temperature for 1 hr. Conjugation (IB-SA) was 129

confirmed by agarose gel electrophoresis (3%). This conjugate (IB-SA) was further 130

conjugated with biotinylated holo-Transferrin (TfB) similarly in a 1:2 ratio. Excess 131

Biocytin (1 nanomole) was added after 1 h to the solution to prevent aggregation. 132

Formation of Transferrin conjugated I-switch (IB-SA-TfB) was confirmed by gel 133

electrophoresis. The presence of Transferrin with the sensor was confirmed by 134

colocalization experiments and by competing out the sensor with Alexa-568 labeled 135

as well as unlabeled Transferrin. 136

137

138

NNANO-08101037 7

139

140

141

142

Supplementary Figure S1. Sequences and modifications used 143

Making the I-switch. The I-switch is composed of three sequences, O1, O2 and O3. 144

O1 and O2 are partially complementary to O3 to form a linear duplex leaving a base 145

gap (shown in red) which acts as a fulcrum to facilitate closing and opening of the 146

construct. Complementary bases in O1 and O3 are shown in green whereas O2 and 147

O3 are represented in blue. i-motif forming sequences are shown as the black 148

overhangs. For fluorescence measurements, O1 and O2 are modified with different 149

fluorophores. O1 was labelled with Alexa 488, Alexa 546 or Bodipy-TMR with a C7 150

linker and O2 was attached similarly to Alexa 647. For conjugation studies, O3 was 151

modified with biotin at 5� terminus with a C6 linker. 152

Name: Sequence

O1 5�-CCCCAACCCCAATACATTTTACGCCTGGTGCC-3�

O2 5�-CCGACCGCAGGATCCTATAAAACCCCAACCCC-3�

O3 5�-TTATAGGATCCTGCGGTCGGAGGCACCAGGCGTAAAATGTA-3�

O-488 5�-Alexa-488-CCCCAACCCCAATACATTTTACGCCTGGTGCC-3�

O-TMR 5�-Bodipy-TMR-CCCCAACCCCAATACATTTTACGCCTGGTGCC-3�

O-647 5�-CCGACCGCAGGATCCTATAAAACCCCAACCCC-Alexa-647-3�

O3-Bio 5�-Biotin-AATTATAGGATCCTGCGGTCGGAGGCACCAGGCGTAAAATGTA-3�

O2/647 O1/488/TMR

O3/Bio




NNANO-08101037 8

CD studies to prove open and closed conformations. 153

154

155

156

157

158

159

160

161

162

163

Supplementary Figure S2. Characterization of I-switch by CD spectroscopy a, CD 164

spectra of I-switch at pH 7.3 (magenta), pH 5 (black) and difference spectra (pH 5 – 165

pH 7.3) is shown in yellow. The cyan trace shows CD of a well known i-motif forming 166

sequence, [d(TC5)]4 showing the peaks of DNA4 i-motif at pH 5.0. Thermal 167

denaturation was carried out on I-switch at pH 5 b, as well as at pH 7.3 c, to 168

demonstrate i-motif formation. Unless mentioned, studies were done using 1 µM I-169

switch in 20 mM phosphate buffer, 100 mM KCl at desired pH. 170

The I-switch shows a positive peak at 278 nm and a negative peak at 248 nm which 171

resembles a B-DNA duplex, whereas spectra taken at pH 5 showed a shift in circular 172

dichroism (CD) spectra with positive and negative peaks at 285 nm and 251 nm 173

A

220 240 260 280 300 320

-4

-2

0

2

4

6

8

10

CD (m

deg)

Wavelength (nm)

pH 5 pH7.3 Difference (pH 5- pH 7.3) d (TC5)

240 260 280 300 320

-20

-10

0

10

20

30

CD

(m

deg)

20°C 25°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C 70°C 75°C 80°C

IncreasingTemperature

Wavelength (nm)240 260 280 300 320

-20

-10

0

10

20

30


CD

(m

deg

)

Wavelength (nm)

b

i

ii

iii

a

civ

v

NNANO-08101037 9

respectively (Fig. S2). The difference spectra between the conformations of the 174

ternary complex at pH 5.0 and pH 7.3 showed a positive peak at 288 nm with a 175

negative peak centred at 262 nm which is in perfect agreement with the characteristic 176

peaks for DNA4 i-motifs. When this difference spectrum was compared with a well-177

characterised i-motif [d(TC5)]4, it perfectly recapitulated the peak pattern 178

characteristics of i-motif. This indicates that at pH 5.0, the ternary assembly of O1, 179

O2 and O3 is composed of duplexed regions as well as an i-motif. 180

In order to confirm this, a thermal melt of the complex at pH 5 was followed by CD 181

(Fig. S2b). 1 μM of I-switch in 20 mM potassium acetate buffer, 100 mM KCl at pH 182

5 was scanned from 20-80°C in steps of 5°C. The spectra were recorded between 330 183

and 235 nm as an average of 8 successive scans. At pH 5, two isochroic points were 184

observed (Fig. S2b), indicating that there were two transitions (one at 50°C and the 185

other at 65°C). At 50°C, the CD signature showed abolition of the i-motif component 186

in the structure, leaving only a signature corresponding to a duplex. At 65°C, this 187

duplex structure too was lost yielding a signature corresponding to single stranded 188

DNA (iii). The difference spectrum between the traces at 25°C and 50°C showed a 189

trace resembling DNA4 i-motifs, confirming that it was indeed the i-motif component 190

that was melted by ~50°C. In contrast, the CD melting experiments done with 1 μM 191

of construct at pH 7.3 (Fig. S2c), showed only one isochroic point corresponding to 192

the melting transition of the duplex (iv) to a single stranded state (v). 193

194

195




NNANO-08101037 8

CD studies to prove open and closed conformations. 153

154

155

156

157

158

159

160

161

162

163

Supplementary Figure S2. Characterization of I-switch by CD spectroscopy a, CD 164

spectra of I-switch at pH 7.3 (magenta), pH 5 (black) and difference spectra (pH 5 – 165

pH 7.3) is shown in yellow. The cyan trace shows CD of a well known i-motif forming 166

sequence, [d(TC5)]4 showing the peaks of DNA4 i-motif at pH 5.0. Thermal 167

denaturation was carried out on I-switch at pH 5 b, as well as at pH 7.3 c, to 168

demonstrate i-motif formation. Unless mentioned, studies were done using 1 µM I-169

switch in 20 mM phosphate buffer, 100 mM KCl at desired pH. 170

The I-switch shows a positive peak at 278 nm and a negative peak at 248 nm which 171

resembles a B-DNA duplex, whereas spectra taken at pH 5 showed a shift in circular 172

dichroism (CD) spectra with positive and negative peaks at 285 nm and 251 nm 173

A

220 240 260 280 300 320

-4

-2

0

2

4

6

8

10

CD (m

deg)

Wavelength (nm)

pH 5 pH7.3 Difference (pH 5- pH 7.3) d (TC5)

240 260 280 300 320

-20

-10

0

10

20

30

CD

(m

deg)

20°C 25°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C 70°C 75°C 80°C


Wavelength (nm)240 260 280 300 320

-20

-10

0

10

20

30


CD

(m

deg

)

Wavelength (nm)

b

i

ii

iii

a

civ

v

NNANO-08101037 9

respectively (Fig. S2). The difference spectra between the conformations of the 174

ternary complex at pH 5.0 and pH 7.3 showed a positive peak at 288 nm with a 175

negative peak centred at 262 nm which is in perfect agreement with the characteristic 176

peaks for DNA4 i-motifs. When this difference spectrum was compared with a well-177

characterised i-motif [d(TC5)]4, it perfectly recapitulated the peak pattern 178

characteristics of i-motif. This indicates that at pH 5.0, the ternary assembly of O1, 179

O2 and O3 is composed of duplexed regions as well as an i-motif. 180

In order to confirm this, a thermal melt of the complex at pH 5 was followed by CD 181

(Fig. S2b). 1 μM of I-switch in 20 mM potassium acetate buffer, 100 mM KCl at pH 182

5 was scanned from 20-80°C in steps of 5°C. The spectra were recorded between 330 183

and 235 nm as an average of 8 successive scans. At pH 5, two isochroic points were 184

observed (Fig. S2b), indicating that there were two transitions (one at 50°C and the 185

other at 65°C). At 50°C, the CD signature showed abolition of the i-motif component 186

in the structure, leaving only a signature corresponding to a duplex. At 65°C, this 187

duplex structure too was lost yielding a signature corresponding to single stranded 188

DNA (iii). The difference spectrum between the traces at 25°C and 50°C showed a 189

trace resembling DNA4 i-motifs, confirming that it was indeed the i-motif component 190

that was melted by ~50°C. In contrast, the CD melting experiments done with 1 μM 191

of construct at pH 7.3 (Fig. S2c), showed only one isochroic point corresponding to 192

the melting transition of the duplex (iv) to a single stranded state (v). 193

194

195




NNANO-08101037 10

196

197

198

199

200

201

202

203

204

205

206

Supplementary Figure S3. Fluorescence Resonance Energy Transfer to 207

demonstrate closed and open states. Fluorescence spectra of the I-switch at pH 5 208

(closed) and pH 7.3 (open) bearing two different FRET pairs. a, I-switch labelled with 209

Alexa 488 and Alexa 647 b, I-switch labelled with Bodipy-TMR and Alexa 647. c,210

Fluorescence melting experiments on I-switch at pH 5 in order to confirm i-motif 211

formation. I-Switch at pH 5 was heated at a rate of 1ºC/min till 75ºC. d, Thermal 212

denaturation profile of the I-switch. Donor intensity (black) at 520 nm and acceptor 213

intensity (red) at 665 nm of the same as a function of temperature. Samples of 80 nM 214

I-switch in 20 mM phosphate buffer, 100 mM KCl at pH 5 were investigated. 215

570 600 630 660 690

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Inte

nsity

(A

U)

Wavelength (nm)

pH 5 pH 7.3

Closed

b

20 30 40 50 60 70 80

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.000

0.002

0.004

0.006

0.008

0.010

Inte

nsi

ty@

520

nm

Temperature (oC)

DonorAcceptor

Inte

nsi

ty @

665

nm

d c

500 550 600 650 700

0.00

0.04

0.08

0.12

0.16

Inte

nsity

(A

U)

Wavelength (nm)

20 OC

30 OC

35 OC

40 OC

45 OC

50 OC

55 OC

60 OC

65 OC

75 OC

500 550 600 650 7000.00

0.05

0.10

Inte

nsi

ty (

AU

)

Wavelength (nm)

pH 5 pH 7.3 pH 5 donor

a




NNANO-08101037 10

196

197

198

199

200

201

202

203

204

205

206

Supplementary Figure S3. Fluorescence Resonance Energy Transfer to 207

demonstrate closed and open states. Fluorescence spectra of the I-switch at pH 5 208

(closed) and pH 7.3 (open) bearing two different FRET pairs. a, I-switch labelled with 209

Alexa 488 and Alexa 647 b, I-switch labelled with Bodipy-TMR and Alexa 647. c,210

Fluorescence melting experiments on I-switch at pH 5 in order to confirm i-motif 211

formation. I-Switch at pH 5 was heated at a rate of 1ºC/min till 75ºC. d, Thermal 212

denaturation profile of the I-switch. Donor intensity (black) at 520 nm and acceptor 213

intensity (red) at 665 nm of the same as a function of temperature. Samples of 80 nM 214

I-switch in 20 mM phosphate buffer, 100 mM KCl at pH 5 were investigated. 215

570 600 630 660 690

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Inte

nsity

(A

U)

Wavelength (nm)

pH 5 pH 7.3

Closed

b

20 30 40 50 60 70 80

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.000

0.002

0.004

0.006

0.008

0.010

Inte

nsi

ty@

520

nm

Temperature (oC)

DonorAcceptor

Inte

nsi

ty @

665

nm

d c

500 550 600 650 700

0.00

0.04

0.08

0.12

0.16

Inte

nsity

(A

U)

Wavelength (nm)

20 OC

30 OC

35 OC

40 OC

45 OC

50 OC

55 OC

60 OC

65 OC

75 OC

500 550 600 650 7000.00

0.05

0.10

Inte

nsi

ty (

AU

)

Wavelength (nm)

pH 5 pH 7.3 pH 5 donor

a

NNANO-08101037 11

FRET and distance measurements. FRET measurements were carried out on I-216

switch composed of labelled DNA strands with Alexa 488 as a donor and Alexa 647 217

as acceptor. This FRET pair has a Förster distance of 5.0 nm.1 Fluorescence spectra 218

were taken at pH 5 and pH 7.3 in phosphate buffer having 100 mM KCl. 54-60% 219

quenching was observed from the closed state to open state giving an interfluorophore 220

distance of 4.6±0.5 nm, which is in good agreement from a coarse grained model 221

taking NMR parameters from d(CCCCAA) i-motif. In order to ensure that the 222

observed FRET was fluorophore independent, another FRET pair was chosen by 223

labelling O1 with Bodipy-TMR and O2 with Alexa-647, which also showed similar 224

energy transfer. When the I-switch incorporated fluorescent labels at a different 225

location, i.e., 5� and 3� termini of O3 with Alexa 546 and Alexa 647 we observed 226

FRET that incorporated the thickness of the duplex arms as well that led to a distance 227

of 7.8±0.5 nm (theoretically predicted distance 7.3 nm). This confirms that in the 228

closed state, the I-switch comprise two duplex arms that are united via their C-rich 229

single stranded overhangs by forming an intramolecular i-motif. 230

CD melts (see earlier section) revealed a lower melting species at 45-50ºC. To 231

confirm that this is due to the melting of an intramolecular i-motif, fluorescence melts 232

were carried out with the sample in which both the overhangs were labelled with 233

dyes. In the closed state via intramolecular i-motif formation, the two dyes come into 234

close proximity (4.6 nm) leading to high FRET. As a function of increasing 235

temperature, thermal denaturation of the i-motif should abolish FRET and therefore 236

donor intensity of the I-switch should increase with a concomitant decrease in 237

acceptor intensity. Figure 3Sd shows the expected sigmoidal increase in donor and 238

concomitant decrease in acceptor fluorescence with a melting temperature of 45ºC 239

consistent with CD data and confirming intramolecular i-motif formation. 240




NNANO-08101037 12

1. Majumdar, D. S., Smirnova, I., Kasho, V, Nir., E., Kong, X., Weiss, S., and 241

Kaback, H. R., Single-molecule FRET reveals sugar-induced conformational 242

dynamics in LacY. Proc. Natl. Acad. Sci. USA. 104, 12640–12645, 2007. 243

Fluorescence Lifetime experiments confirms FRET 244

Donor fluorescence intensity decays for donor-only (D) labelled (Alexa 488) and 245

donor-acceptor (DA) labelled (Alexa 488 as donor and Alexa 647 as acceptor) 246

construct were obtained from TCSPC experimental setup. To quantify donor 247

fluorescent lifetime, intensity decays were fitted to a bi-exponential decay model 248

1 2( ) ( / )0 1 2

t tI I A e A e� �� by an iterative reconvolution procedure using a 249

Levenberg-Marquardt minimization algorithm, where 0I is initial fluorescence 250

intensity; 1 2,A A are the normalized amplitude of individual components; and 1 2,� � are 251

the lifetimes of each components. Fluorescence decays were considered well fit when 252

three criteria were met: a) reduced 2� was less than 1.2, b) residuals were evenly 253

distributed across the full extent of the data, and c) visual inspection ensured that the 254

fit accurately described the decay profile. A representative time resolved fluorescence 255

intensity decay and fit from donor-only sample is shown in Figure S4a. 256

Parameters such as amplitude 1 2( , )A A and decay time constants 1 2( , )� � were 257

obtained after satisfying above criteria of fitting and the average lifetime was 258

calculated using the formula 1 1 2 2

1 2

( ) ( )av

A AA A� ��

��

(Where ��+�� was normalized 259

to 1) and listed in the table below. Although, at pH 7.3 D-labelled construct showed 260

an average lifetime of 3.77 ns which marginally decreased to 3.5 ns at pH 6, at pH 5 it 261

dropped to 3.0 ns that can be explained by donor quenching due to i-motif 262




NNANO-08101037 12

1. Majumdar, D. S., Smirnova, I., Kasho, V, Nir., E., Kong, X., Weiss, S., and 241

Kaback, H. R., Single-molecule FRET reveals sugar-induced conformational 242

dynamics in LacY. Proc. Natl. Acad. Sci. USA. 104, 12640–12645, 2007. 243

Fluorescence Lifetime experiments confirms FRET 244

Donor fluorescence intensity decays for donor-only (D) labelled (Alexa 488) and 245

donor-acceptor (DA) labelled (Alexa 488 as donor and Alexa 647 as acceptor) 246

construct were obtained from TCSPC experimental setup. To quantify donor 247

fluorescent lifetime, intensity decays were fitted to a bi-exponential decay model 248

1 2( ) ( / )0 1 2

t tI I A e A e� �� by an iterative reconvolution procedure using a 249

Levenberg-Marquardt minimization algorithm, where 0I is initial fluorescence 250

intensity; 1 2,A A are the normalized amplitude of individual components; and 1 2,� � are 251

the lifetimes of each components. Fluorescence decays were considered well fit when 252

three criteria were met: a) reduced 2� was less than 1.2, b) residuals were evenly 253

distributed across the full extent of the data, and c) visual inspection ensured that the 254

fit accurately described the decay profile. A representative time resolved fluorescence 255

intensity decay and fit from donor-only sample is shown in Figure S4a. 256

Parameters such as amplitude 1 2( , )A A and decay time constants 1 2( , )� � were 257

obtained after satisfying above criteria of fitting and the average lifetime was 258

calculated using the formula 1 1 2 2

1 2

( ) ( )av

A AA A� ��

��

(Where ��+�� was normalized 259

to 1) and listed in the table below. Although, at pH 7.3 D-labelled construct showed 260

an average lifetime of 3.77 ns which marginally decreased to 3.5 ns at pH 6, at pH 5 it 261

dropped to 3.0 ns that can be explained by donor quenching due to i-motif 262

NNANO-08101037 13

formaition1. In contrast, at both pH (6 and 5) donor lifetime exhibited dramatic 263

decrease in DA-labelled (2.7 ns and 2.1 ns) sample compared to D-labelled (3.5 ns 264

and 3.5 ns) sample respectively (Fig. S4b). It is also evident that at both pH 6 and 5, a 265

faster decay component appears which can only be fitted with a third exponent. 266

Appearance of this faster component can be explained as a result of FRET 2. Average 267

lifetime extracted from the all decay curves are summarized in table 2 below. 268

1. Mergny, J. L. Fluorescence energy transfer as a probe for tetraplex formation: 269

the i-motif. Biochemistry, 3, 1573-81, 1999. 270

2. Mountford, C. P. et.al., Time-Resolved FRET and FLIM of Four-way DNA 271

Junctions. Journal of Fluorescence, 16, 839–845, 2006 272

273 274

275

276

277

278

279

Supplementary Figure S4. Time resolved Fluorescence spectra of differently 280

labelled I-switch assemblies at different pH. Lifetime decay of a, Donor only labelled 281

and b, Dual labelled (Alexa-488/647) I-switch at indicated pH was measured using 282

multiphoton excitation. 5 µM I-switch was diluted with phosphate buffer of appropriate 283

pH containing 100 mM KCl before acquisition. 284

0.5

1

0

0

0

0 20

Nor

mal

ized

cou

nts pH 5.0

pH 6.0 pH 6.5 pH 7.3

Time (ns)

0.5

1

00

0 20

0Nor

mal

ized

cou

nts

pH 5.0 pH 6.0 pH 6.5 pH 7.3

Time (ns)

a b




NNANO-08101037 14

Supplementary Table 2(A) and (B). Summary of the average lifetime of dual 285

labelled I-switch (Alexa 488-Alexa 647) as well as donor-only controls in vitro286

and inside endosomes respectively.287

2A 2B 288

289 290

291

292

293

294

295

296

pH

Donor Only

(�d) (ns) ± SD

Dual labelled

(�da) (ns) ± SD

5.0 3.021 ± 0.003 2.182 ± 0.094

6.0 3.584 ± 0.013 2.728 ± 0.035

6.5 3.737 ± 0.015 3.273 ± 0.012

7.2 3.777 ± 0.027 3.506 ± 0.015

* After clamping the pH at desired value using Nigericin.

pH*

Timepoint inside

endosome (min)

Dual labelled

(�da) (ns)

Dual labeled*

(�da) (ns) ± SD

7 - - 1.173±0.035

5.5 30 - -

5 120 0.8 0.735±0.067




NNANO-08101037 14

Supplementary Table 2(A) and (B). Summary of the average lifetime of dual 285

labelled I-switch (Alexa 488-Alexa 647) as well as donor-only controls in vitro286

and inside endosomes respectively.287

2A 2B 288

289 290

291

292

293

294

295

296

pH

Donor Only

(�d) (ns) ± SD

Dual labelled

(�da) (ns) ± SD

5.0 3.021 ± 0.003 2.182 ± 0.094

6.0 3.584 ± 0.013 2.728 ± 0.035

6.5 3.737 ± 0.015 3.273 ± 0.012

7.2 3.777 ± 0.027 3.506 ± 0.015

* After clamping the pH at desired value using Nigericin.

pH*

Timepoint inside

endosome (min)

Dual labelled

(�da) (ns)

Dual labeled*

(�da) (ns) ± SD

7 - - 1.173±0.035

5.5 30 - -

5 120 0.8 0.735±0.067

NNANO-08101037 15

In vitro pH profile of Bodipy-TMR/Alexa 647 labeled I-switch. 297

298

299

300

301

302

303

Supplementary Figure S5. I-switch as a versatile probe. In vitro calibration curve for 304

Bodipy- TMR and Alexa 647 labelled I-switch determined as described in main text. 305

a, Fluorescence intensity plotted as a function of pH to generate standard curve b,.306

Comparison of this pH profile with a different FRET pair of Alexa 488/647 on the I-307

switch. Data is represented as a mean of four individual experiments ± s.e.m plotted 308

as a function of pH. 309

To demonstrate that the I-switch is a ratiometric probe of pH that is independent of 310

the FRET pair, we labelled the I-switch with two different FRET pairs Bodipy-311

TMR/Alexa 647 as shown in (Fig. S5a) and comparison with Alexa 488/647 FRET 312

pair which is shown in (b). 80 nM I-switch labelled with Bodipy TMR and Alexa 647 313

was taken in buffers ranging from pH 4 to 7.3 containing 100 mM KCl. Bodipy TMR 314

was excited at 543 nm and spectra was recorded from 560 to 750 nm. D/A curve as a 315

function of pH was generated from the ratio of donor (579 nm) to acceptor (665 nm) 316

intensities at the given pH values. Fold increase for this FRET pair, calculated as 317

described in main text, was found to be 3.5 while the fold increase in case of Alexa 318

4 5 6 7

0.4

0.8

1.2

Don

or/A

ccep

tor

pH

BodipyTMR/A 647 A 488/A 647

a b

560 580 600 620 640 660 680 700 720 740

0.00

0.05

0.10

0.15

0.20

0.25

0.30In

tens

ity (

AU

)

Wavelength (nm)

4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.3




NNANO-08101037 16

488/Alexa-647 pair was found to be 5.5. Due to higher dynamic range, Alexa- 319

488/647 was used for further studies unless otherwise specified. Importantly the 320

regime of pH sensitivity in both cases is identical indicating that the transition is 321

completely fluorophore independent. 322

323




NNANO-08101037 16

488/Alexa-647 pair was found to be 5.5. Due to higher dynamic range, Alexa- 319

488/647 was used for further studies unless otherwise specified. Importantly the 320

regime of pH sensitivity in both cases is identical indicating that the transition is 321

completely fluorophore independent. 322

323

NNANO-08101037 17

Spatiotemporal pH mapping during endocytosis 324

325

326

327

328

329

330

331

332

333

Supplementary Figure S6. Receptor mediated pathway during endocytosis. This is 334

a schematic of the internalisation of I-switch when tagged with Transferrin. At early 335

time points, early endosomes are Rab-5 positive and relatively less acidic. These 336

progressively mature to lysosomes in about 2 hrs which is much more acidic and 337

stained by LysoTracker™. 338

Temporal pH mapping during endocytosis. There are several mechanisms of 339

endocytosis and of specific interest to the I-switch is the receptor mediated 340

endocytotic pathway. When a specific ligand binds with its receptor, the ligand is 341

internalized by the cell through the formation of a vesicle termed as the early 342

endosome. The early endosome has a pH 6 to 6.5. Rab 5 is a GTPase which is 343

localised in early endosomes and is therefore used as a molecular marker for an early 344




NNANO-08101037 18

endosome. The early endosome gradually matures to late endosome and lysosome 345

which is identified by LysoTracker ™ red staining. 346

347

The I-switch specifically marks the ALBR endocytic pathway 348

It is known that cargo entering cells via endocytic pathways such as the anionic 349

ligand binding receptor (ALBR) pathway fuse with FITC dextran labelled endosomes 350

at the indicated chase times. Competition experiments with maleylated BSA (mBSA) 351

that is known to adopt the ALBR mediated endocytic route in hemocytes indicated 352

that the I-switch exclusively marks out endosomes along this pathway. Cells were 353

pulsed with a mixture of I-switch (Alexa 647) (80 nM) and mBSA (0.8 mg/ml), 354

chased for 5 min and fixed. Total fluorescence of the cells was measured and 355

normalized with respect to the fluorescence in untreated cells and is presented as 356

fraction of I-switch internalised. Figure S7b shows that the I-switch is clearly 357

competed out in the presence of excess mBSA. Till the 2 h time point, the I-switch is 358

in punctate endosomal structures indicating that it is stable inside cells on these time 359

scales (Fig. S7a). This was further confirmed by fluorescence lifetime measurements 360

of the donor on doubly-labelled I-switch (See Table 2B) inside endosomes. When the 361

lifetime of donor in dual labelled I-switch was measured inside endosomes at t = 2h, 362

these showed characteristic low donor lifetimes (0.8 ns). Upon pH clamping of these 363

endosomes at pH 5, similar donor lifetimes were observed (0.74 ns) indicating that at t 364

= 2h, both fluorophores on the I-switch were within FRETting distance. Upon 365

clamping endosomes at pH 7, when the donor was out of FRETting distance, 366

distinctly higher lifetime of 1.2 ns was observed. 367

368




NNANO-08101037 18

endosome. The early endosome gradually matures to late endosome and lysosome 345

which is identified by LysoTracker ™ red staining. 346

347

The I-switch specifically marks the ALBR endocytic pathway 348

It is known that cargo entering cells via endocytic pathways such as the anionic 349

ligand binding receptor (ALBR) pathway fuse with FITC dextran labelled endosomes 350

at the indicated chase times. Competition experiments with maleylated BSA (mBSA) 351

that is known to adopt the ALBR mediated endocytic route in hemocytes indicated 352

that the I-switch exclusively marks out endosomes along this pathway. Cells were 353

pulsed with a mixture of I-switch (Alexa 647) (80 nM) and mBSA (0.8 mg/ml), 354

chased for 5 min and fixed. Total fluorescence of the cells was measured and 355

normalized with respect to the fluorescence in untreated cells and is presented as 356

fraction of I-switch internalised. Figure S7b shows that the I-switch is clearly 357

competed out in the presence of excess mBSA. Till the 2 h time point, the I-switch is 358

in punctate endosomal structures indicating that it is stable inside cells on these time 359

scales (Fig. S7a). This was further confirmed by fluorescence lifetime measurements 360

of the donor on doubly-labelled I-switch (See Table 2B) inside endosomes. When the 361

lifetime of donor in dual labelled I-switch was measured inside endosomes at t = 2h, 362

these showed characteristic low donor lifetimes (0.8 ns). Upon pH clamping of these 363

endosomes at pH 5, similar donor lifetimes were observed (0.74 ns) indicating that at t 364

= 2h, both fluorophores on the I-switch were within FRETting distance. Upon 365

clamping endosomes at pH 7, when the donor was out of FRETting distance, 366

distinctly higher lifetime of 1.2 ns was observed. 367

368

NNANO-08101037 19

*

a

369

370

371

372

373

374

375

Supplementary Figure S7. I-switch is endocytosed via the anionic ligand binding 376

receptor (ALBR) pathway. a, Alexa 488-647 labelled I-switch pulsed for 5 min, 377

chased for 2 h and imaged shown colocalization of two colours in distinct punctate 378

endosomes. b, Quantification of I-switch internalization by Drosophila hemocytes in 379

the presence and absence of excess of maleylated BSA (mBSA, 100µg/ml) that 380

saturates ALB receptors. I-switch internalization was quantified in terms of the total 381

fluorescence intensity (at Alexa-647 channel) of cells pulsed with I-switch in the 382

presence and absence of excess unlabeled mBSA. The contribution from 383

autofluorescence is also shown. Scale Bar: 5 µm, inset: 1 µm. 384

385

386

387

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n of

i-sw

itch

i nte

rnal

ized

No mBSA + mBSA Autofluorescence

b




NNANO-08101037 20

388

Spatiotemporal pH mapping during endocytosis. 389

390

391

392

393

394

Supplementary Figure S8. Temporal pH mapping during endocytosis. a, Enlarged 395

view of intracellular standard curve to show measured pH during endocytosis. Blue 396

curve represents calibration curve obtained by adding Nigericin and red squares are 397

mean D/A (± s.e.m) values of endosomes after a 5 min pulse, with a chase of 5 min, 398

1h and 2 h. b, Mean D/A of endosomes at different chase times (t= 5, 60 and 120 399

min) were converted to their respective mean pH values and plotted as a function of 400

chase time (t). Error bar: Mean ± s.e.m 401

Nigericin is a potassium-H+ antiporter which exchanges protons and depletes pH 402

gradients inside cells, making endosomal pH the same as the pH of the external buffer 403

added to the cells. D/A ratio of endosomes in Drosophila hemocytes at desired pH 404

were measured and plotted with respect to pH. Intracellularly, a sigmoidal increase in 405

D/A ratio curve was obtained with a 5 fold increase from closed to open states (Fig. 406

2a, maintext). When this D/A curve was overlayed (2A) with the in vitro curve it 407

recapitulated qualitatively and quantitatively its closing and opening characteristics 408

inside cells. D/A value of each endosome was collected and presented in a histogram 409

(Fig. 3b, main text) and from that mean D/A was calculated at 5 min,1 h and 2 h, 410

0 20 40 60 80 100 120

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

pH

Time (min)

Mean D/A

b

Mean pH

5.0 5.5 6.0

1.0

1.5

Don

or/

Acc

epto

r

pH

Measured pH clamping

a




NNANO-08101037 20

388

Spatiotemporal pH mapping during endocytosis. 389

390

391

392

393

394

Supplementary Figure S8. Temporal pH mapping during endocytosis. a, Enlarged 395

view of intracellular standard curve to show measured pH during endocytosis. Blue 396

curve represents calibration curve obtained by adding Nigericin and red squares are 397

mean D/A (± s.e.m) values of endosomes after a 5 min pulse, with a chase of 5 min, 398

1h and 2 h. b, Mean D/A of endosomes at different chase times (t= 5, 60 and 120 399

min) were converted to their respective mean pH values and plotted as a function of 400

chase time (t). Error bar: Mean ± s.e.m 401

Nigericin is a potassium-H+ antiporter which exchanges protons and depletes pH 402

gradients inside cells, making endosomal pH the same as the pH of the external buffer 403

added to the cells. D/A ratio of endosomes in Drosophila hemocytes at desired pH 404

were measured and plotted with respect to pH. Intracellularly, a sigmoidal increase in 405

D/A ratio curve was obtained with a 5 fold increase from closed to open states (Fig. 406

2a, maintext). When this D/A curve was overlayed (2A) with the in vitro curve it 407

recapitulated qualitatively and quantitatively its closing and opening characteristics 408

inside cells. D/A value of each endosome was collected and presented in a histogram 409

(Fig. 3b, main text) and from that mean D/A was calculated at 5 min,1 h and 2 h, 410

0 20 40 60 80 100 120

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

pH

Time (min)

Mean D/A

b

Mean pH

5.0 5.5 6.0

1.0

1.5

Don

or/

Acc

epto

r

pH

Measured pH clamping

a

NNANO-08101037 21

compared with the standard curve in order to obtain pH at early, late and lysosomes 411

respectively (Fig. S8b). 412

413

414

415

416




NNANO-08101037 22

Conjugation of I-switch with a protein of interest. 417

418

419

420

421

422

423

424

425

426

Supplementary Figure S9. Electrophoretic mobility shift assay showing 427

complexation of Streptavidin conjugated I-switch (IB-SA) with Biotinylated Transferrin 428

(TfB).429

Formation of IB-SA and IB-SA-TfB conjugate was confirmed by 3% Agarose gel. Upon 430

Streptavidin (SA) conjugation, the band corresponding to the I-switch shifts to a 431

slower migrating species (IB-SA). To a solution of IB-SA (25 pmole) Biotinylated 432

human holo-Transferrin (TfB) was added in varying ratios, incubated at room 433

temperature for 1 h and then excess Biocytin was added to prevent aggregation, which 434

was further confirmed by gel electrophoresis. With increasing TfB, the gradual 435

increase of a slow moving band (IB-SA-TfB) was observed near the well with a 436

concomitant disappearance of IB-SA (Fig. S9). Best results were obtained when ratio of 437

IB: SA: TfB was 1:1:2. Further increase of TfB led to turbidity. 438

A

IB-SA-TfB

IB-SA

IB




NNANO-08101037 22

Conjugation of I-switch with a protein of interest. 417

418

419

420

421

422

423

424

425

426

Supplementary Figure S9. Electrophoretic mobility shift assay showing 427

complexation of Streptavidin conjugated I-switch (IB-SA) with Biotinylated Transferrin 428

(TfB).429

Formation of IB-SA and IB-SA-TfB conjugate was confirmed by 3% Agarose gel. Upon 430

Streptavidin (SA) conjugation, the band corresponding to the I-switch shifts to a 431

slower migrating species (IB-SA). To a solution of IB-SA (25 pmole) Biotinylated 432

human holo-Transferrin (TfB) was added in varying ratios, incubated at room 433

temperature for 1 h and then excess Biocytin was added to prevent aggregation, which 434

was further confirmed by gel electrophoresis. With increasing TfB, the gradual 435

increase of a slow moving band (IB-SA-TfB) was observed near the well with a 436

concomitant disappearance of IB-SA (Fig. S9). Best results were obtained when ratio of 437

IB: SA: TfB was 1:1:2. Further increase of TfB led to turbidity. 438

A

IB-SA-TfB

IB-SA

IB

NNANO-08101037 23

Size exclusion chromatography (SEC) to assess homogeneity in TfB conjugated I-439

switch. 440

Size exclusion chromatography was performed on a Shimadzu HPLC system using an 441

SEC column BioSep-SEC-S3000 (Phenomenex, with 5 μm beads, column 442

dimensions: 300mm � 4.6 mm, total column volume (Vc) = 4.98mL, measured void 443

volume (Vo) = 1.9 mL). It is a hydrophilic bonded Silica based column with a pore 444

size of 29 nm, and has exclusion limits of 15 kDa - 700 kDa. IB, IB-SA and IB-SA-TfB 445

was prepared as described earlier, diluted to 1μM concentration with PBS prior to 446

injection and eluted with an isocratic flow of PBS over 30 mins. For IB, SA and IB-SA 447

absorbance at 260 nm was followed whereas Transferrin and its conjugates were 448

followed with at the 475 nm absorbance characteristic of transferrin to determine 449

heterogeneity in the IB-SA-TfB conjugate. 450

Pure IB and pure SA showed single peaks with a retention time (Rt) of 6.2 min and 7 451

min respectively (Fig. S10a,b). In contrast, a mixture of 1:1 IB:SA showed the 452

appearance of a new peak (Rt: 5.2 min) that is assigned to 1:1 IB-SA. In addition, peaks 453

corresponding to free IB and free SA respectively are also seen (Fig. S10c). Notably 454

there were no other peaks with a low Rt indicating that the complex formed between 455

IB-SA is 1:1. This is consistent with gel electrophoresis data where only a single SA-456

complexed band was observed. 457

Pure TfB showed a predominant single peak around 6.6 min with a very minor peak 458

near 8.3 min (indicated by *) that could correspond to some impurity in the procured 459

sample (Fig. S10d). When IB-SA is added to TfB in a 1:2 ratio it could result in 460

multiple TfB per IB-SA. However, the SEC chromatogram at 475 nm (a wavelength 461

where only TfB is active) shows predominantly only a uniquely-sized species of IB-SA 462

complexed TfB. If a heterogeneous population of more than one TfB resulted, one 463

would expect multiple peaks for this wavelength. However, Figure S10d shows a 464




NNANO-08101037 24

chromatogram that has only two peaks, one corresponding to uncomplexed TfB and 465

another significantly faster than either of the starting components (4.3 min) which we 466

assign as IB-SA-TfB. There are no other significant populations corresponding to more 467

than one TfB. Such species must be resolvable on the SEC given that the m.w. of TfB 468

is ~80 kDa, and indicates that the current protocol results in the formation of an 469

homogeneous IB-SA-TfB complex. The small shoulder in the peak of IB-SA-TfB 470

(indicated by **, Fig. S10c, Rt = 5.2 min) probably originates from the complexation 471

of IB-SA with the small impurity (*, Fig.s10c Rt = 8.3 min) in the TfB sample. 472

473

474

475

476

477

478

479

Supplementary Figure S10. Size exclusion chromatography (SEC) established 480

complex stoichiometry. SEC chromatogram of (a) Streptavidin (SA) and I-switch (IB)481

showing single peaks whereas 1:1 complex of SA and IB showing two peaks of IB-SA482

and IB correspondingly (c). 50 µL of 1 µM I-switch or complex was injected and 483

separated using an isocratic flow of PBS with a flow rate of 0.5 ml/min. Absorbance 484

at 260 nm was followed over time. (d) Chromatogram showing free TfB and when TfB485

was added to IB-SA in a 2:1 ratio, a new peak arises due to formation of IB-SA-TfB (e). 486

Free TfB

IB

TfB

IB-SA-TfB

IB-SA IB-SA

Free IB and SA

d

e

c

b

a

IB-SA-TfB

IB

Vo

Vo

Vo

Vo

Vo

*

***




NNANO-08101037 24

chromatogram that has only two peaks, one corresponding to uncomplexed TfB and 465

another significantly faster than either of the starting components (4.3 min) which we 466

assign as IB-SA-TfB. There are no other significant populations corresponding to more 467

than one TfB. Such species must be resolvable on the SEC given that the m.w. of TfB 468

is ~80 kDa, and indicates that the current protocol results in the formation of an 469

homogeneous IB-SA-TfB complex. The small shoulder in the peak of IB-SA-TfB 470

(indicated by **, Fig. S10c, Rt = 5.2 min) probably originates from the complexation 471

of IB-SA with the small impurity (*, Fig.s10c Rt = 8.3 min) in the TfB sample. 472

473

474

475

476

477

478

479

Supplementary Figure S10. Size exclusion chromatography (SEC) established 480

complex stoichiometry. SEC chromatogram of (a) Streptavidin (SA) and I-switch (IB)481

showing single peaks whereas 1:1 complex of SA and IB showing two peaks of IB-SA482

and IB correspondingly (c). 50 µL of 1 µM I-switch or complex was injected and 483

separated using an isocratic flow of PBS with a flow rate of 0.5 ml/min. Absorbance 484

at 260 nm was followed over time. (d) Chromatogram showing free TfB and when TfB485

was added to IB-SA in a 2:1 ratio, a new peak arises due to formation of IB-SA-TfB (e). 486

Free TfB

IB

TfB

IB-SA-TfB

IB-SA IB-SA

Free IB and SA

d

e

c

b

a

IB-SA-TfB

IB

Vo

Vo

Vo

Vo

Vo

*

***

NNANO-08101037 25

Transferrin has a 475 nm absorbance peaks and was followed to probe number of 487

bound transferrins on a single IB-SA complex. 488

I-switch tagged to Transferrin is internalized by RME pathway. Internalization of 489

IB-SA-TfB via transferrin receptor pathway was further confirmed by competition 490

studies with excess unlabeled Transferrin. Briefly, human holo- Transferrin, Tf (100 491

μg) was bound with transferrin receptor expressed in Drosophila SR+ cells on ice for 492

15 min and then chased for another 15 min in presence of I-switchylated transferrin 493

(IB-SA-TfB), stripped to remove any surface bound probe, fixed and imaged in a 494

widefield microscope using a 20x objective. 100 cells per view were quantified. Total 495

intensity of each cell in Alexa-647 channel was presented as a mean ± s.e.m of two 496

different experiments. 497

498

. 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513

Supplementary Figure S11. Competition studies with unlabelled Transferrin (Tf) 514

demonstrates that I-switch conjugated to Transferrin (IB-SA-TfB) is internalized via 515

transferrin receptors. + Transferrin indicates intensity of cells pulsed with IB-SA-TfB in 516

presence of Tf, –Transferrin is cells pulsed with IB-SA-TfB alone whereas control cells 517

were pulsed with I-switch lacking biotinylated Transferrin (IB-SA). 518

+ Transferrin - Transferrin Control0

200

400

600

800

1,000

Inte

nsity

@ 6

47

nm

Intensity



supplementary information doi: 10.1038/nnano.2009.83NNANO-08101037 26

Supplementary Table 3: A comparison of different fluorescent pH sensors used for various biological applications and their limitations.

Name pKa Working regime

Sensitivity at (pH 6-7) Advantages Disadvantages Ref

FITC 6.5 5-8 ~2 fold Highest sensitivity at pH above

7Photobleaching Decrease in fluorescence with acidification

1-2

BCECF 6.98 6-8 ~2 fold Ideal to study pH 7-8.

Ratiometric Intracellular hydrolysis of the ester bond limiting the dynamic range of fluorescence ratio determination

3-4

Carboxy-SNARF-1

7.3-7.5 6.4-8 ~2.5 pH dependent dual emission High S/N ratio for ratiometric imaging

Low fluorescence quantum yield High pKa limits biological application below pH 7.

5-6

Ecliptic phluorin

7.1 5-8 ~ 2 fold Genetically encoded Can be made ratiometric

Fluorescence is quenched at acidic pH – therefore not visible below pH 6.0.Single wavelength probe (i.e. GFP)

7-8

Oregon green 488

4.7 4.0-5.0 3 fold (from pH 4-5)

Best suited to study lysosomal pH

Only highly acidic organelles can only be studied

9

I-switch

6.4 5.5-7 > 3.6 fold Bright, photostable, easy to use. FRET-based pH sensor: Not limited by fluorophore wavelengths – several FRET pairs can be used. Highest dynamic range from pH 5.5 to 7

Not possible to study pH regimes pH<5-6.8<pH Reduced sensitivity between pH 5-5.5 (only ~1.5-1.8 fold increase)



supplementary informationdoi: 10.1038/nnano.2009.83NNANO-08101037 26

Supplementary Table 3: A comparison of different fluorescent pH sensors used for various biological applications and their limitations.

Name pKa Working regime

Sensitivity at (pH 6-7) Advantages Disadvantages Ref

FITC 6.5 5-8 ~2 fold Highest sensitivity at pH above

7Photobleaching Decrease in fluorescence with acidification

1-2

BCECF 6.98 6-8 ~2 fold Ideal to study pH 7-8.

Ratiometric Intracellular hydrolysis of the ester bond limiting the dynamic range of fluorescence ratio determination

3-4

Carboxy-SNARF-1

7.3-7.5 6.4-8 ~2.5 pH dependent dual emission High S/N ratio for ratiometric imaging

Low fluorescence quantum yield High pKa limits biological application below pH 7.

5-6

Ecliptic phluorin

7.1 5-8 ~ 2 fold Genetically encoded Can be made ratiometric

Fluorescence is quenched at acidic pH – therefore not visible below pH 6.0.Single wavelength probe (i.e. GFP)

7-8

Oregon green 488

4.7 4.0-5.0 3 fold (from pH 4-5)

Best suited to study lysosomal pH

Only highly acidic organelles can only be studied

9

I-switch

6.4 5.5-7 > 3.6 fold Bright, photostable, easy to use. FRET-based pH sensor: Not limited by fluorophore wavelengths – several FRET pairs can be used. Highest dynamic range from pH 5.5 to 7

Not possible to study pH regimes pH<5-6.8<pH Reduced sensitivity between pH 5-5.5 (only ~1.5-1.8 fold increase)

NNANO-08101037 27

Supplementary References:

1. Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralysosomal

pH in living cells and the perturbation of pH by various agents. Proc. Natl.

Acad. Sci. USA. 75, 3327-3331 (1978).

2. Lanz, E., Gregor, M., Slavík, J., & Kotyk, A. Use of FITC as a Fluorescent

Probe for Intracellular pH Measurement. Journal of Fluorescence 7, 317-

319,(1997).

3. Gan, B. S., Krump, E., Shrode, L. D., & Grinstein, S. Loading pyranine via

purinergic receptors or hypotonic stress for measurement of cytosolic pH by

imaging. Am J Physiol Cell Physiol 275, 1158-1166, (1998).

4. Grant, R. & Acosta, D. Ratiometric measurement of intracellular pH of cultured

cells with BCECF in a fluorescence multi-well plate reader. In Vitro Cell Dev.

Biol.--Animal 33, 256-260, (1997).

5. Buckler, J. & Vaughan-Jones, R. D. Application of a new pH-sensitive

fluorophore Carboxy-SNARF-1 for intracellular pH measurement in small,

isolated cells. European Journal of Physiology 417, 234- 239, (1990).

6. Seksek. O., & Bolard, J. Nuclear pH gradient in mammalian cells revealed by

Laser microspectrofluorimetry. Journal of Cell Science 109, 257-262 (1996).

7. Miesenböck, G., De Angelis, D., & Rothman, J. E. Visualizing secretion and

synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394,

192-195, (1998).

8. Sankaranarayanan, S., De Angelis, D., Rothman, J. E., & Ryan, T. A. The Use

of pHluorins for Optical Measurements of Presynaptic Activity. Biophys J 79,

2199-2208, (2000).

9. Porte, F., Liautard, J-P & Köhler, S., Early acidification of phagosomes

containing Brucella suis is essential for intracellular survival in murine

macrophages. Infect Immun. 67, 4041–4047, (1999).


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