SUPPORTING INFORMATION Visualization and quantification of microrna in a single cell using atomic force microscopy Hyunseo Koo, Ikbum Park, Yoonhee Lee, Hyun Jin Kim, Jung Hoon Jung, Joo Han Lee, Youngkyu Kim *, Joung-Hun Kim * and Joon Won Park * * e-mail: ynq.kim@gmail.com; joungkim@postech.ac.kr; jwpark@postech.ac.kr Supplementary Experimental Procedures Supplementary Figures 1 11 Supplementary Tables 1, 2 S1
Supplementary Experimental Procedures Purification of GST-fused HBD. GST-fused HBD was cloned into the pgex-4t-1 construct and expressed in BL21 (DE3) cells. The cells were cultured in LB medium containing carbenicillin, and protein expression was induced by the addition of.2 mm IPTG at 37 C for 4 h. The cultured cells were re-suspended in lysis buffer (5 mm Tris-HCl, 2 mm NaCl,.5% Tween 2 (v/v), 1. mm DTT, 2. mm EDTA, ph 8.) containing a protease inhibitor cocktail (Roche) and were lysed by sonication. The lysate was centrifuged at 25,g at 4 C for 25 min, and the supernatant was loaded onto a GSH-agarose resin. GST-HBD protein was eluted with 5 mm Tris buffer containing 1. mm EDTA and 15 mm reduced glutathione (ph 9.5). The eluted proteins were concentrated in storage buffer (5 mm Tris-HCl, 2 mm NaCl, 2 mm EDTA, ph 8.) using a centrifugal filter (1 kd cut off, EMD Millipore) and confirmed by SDS-PAGE. Neuron culture. C57BL/6 mouse hippocampal neurons were dissected from postnatal day 1 pups and cultured on poly-l-lysine (Sigma)-coated plates and cover slips. Neurons were plated in neurobasal medium (Invitrogen) supplemented with 2.% B27 (Invitrogen), 1.% GlutaMAX TM (Invitrogen), 2.% fetal bovine serum (Hyclone) and 1.% penicillin/streptomycin (Invitrogen) (v/v) in a humidified 5% CO 2 /95% O 2 incubator at 37 C. After 6 8 h, the medium was replaced with serum-free conditioned neurobasal medium. At DIV7, a group of neurons was treated with KCl (final concentration 4 mm) for 2 h before cell lysis, aspiration or fixation. N2a culture. N2a cells were cultured on poly-l-lysine-coated cover slips. Cells were grown in 44.5% DMEM (Welgene) and 44.5% Opti-MEM (GIBCO) supplemented with 1% fetal bovine serum (Hyclone) and 1.% penicillin/streptomycin (Invitrogen) (v/v) in a humidified 5% CO 2 /95% O 2 incubator at 37 C. S2
Single-cell aspiration. After KCl treatment, hippocampal neurons were moved into a whole-cell patch clamp setup. Neurons were continuously perfused with extracellular solution (119 mm NaCl, 2.5 mm KCl, 2. mm MgSO 4, 1.25 mm NaH 2 PO 4, 26 mm NaHCO 3, 2.5 mm CaCl 2, and 1 mm dextrose, ph 7.4) at room temperature, and a whole-cell patch clamp was generated using borosilicate micropipettes (3 5 MΩ) filled with 3 5 µl internal solution (135 mm KCl, 1 mm NaCl, 2 mm MgCl 2,.5 mm EGTA, and 1 mm HEPES, ph 7.2 adjusted with KOH). SUPERase-In (2. µl ml -1, Ambion) was also included in the internal solution to inhibit RNase activity. After a whole-cell patch clamp recording was obtained with a Multiclamp 7B amplifier (Molecular Devices), the cell was aspirated by applying negative pressure with a connected 5-ml glass syringe. The aspiration step took no longer than 5 min, and the recording electrode was cleaned after each aspiration to prevent cell-to-cell contamination. Cells exhibiting a >1 pa change in holding current during the aspiration were discarded. 1 The aspirated cytoplasm was immediately delivered into an RNase-free PCR tube containing 7. µl QIAzol lysis solution by breaking the tip and applying positive pressure. To minimize the effect of RNases, all glassware, including micropipettes, were incubated at 17 C overnight, and other equipment was cleaned with RNaseZap (Ambion). Total RNA extraction. To isolate RNA from neurons in the culture plate, neurons were rinsed with ice-cold Dulbecco s PBS, and 5 µl QIAzol lysis solution was added. Cell lysis and total RNA extraction were performed for samples collected from the culture plate or single-cell aspiration using a mirneasy Micro Kit (Qiagen) according to the manufacturer s protocol. The concentration (w/v) and purity of RNA extracted from neurons in a culture plate were S3
determined by measuring the absorbance at 23, 26 and 28 nm with an ND-1 spectrophotometer (NanoDrop Technologies). Quantitative RT-PCR. Total RNA samples and synthetic mir-134 were polyadenylated and reverse transcribed at 37 C for 1 h using a miscript II RT Kit (Qiagen). cdna made from the total RNA samples was diluted 2-fold for the PCR reactions. qpcr analysis was performed in triplicate on a LightCycler 2. (Roche) using a miscript SYBR Green PCR Kit and mir-134- specific primers (Qiagen), and the data were analyzed using an automatic cycle threshold setting. A standard curve was generated at each reaction from the cdna of synthetic mir-134 prepared at five different concentrations (1.2 1 8 1.2 1 4 copies in the PCR reaction solution). The copy number of mir-134 in a single neuron was calculated assuming a total RNA mass of 2 pg per cell. RNase H treatment. The probe spots or fixed cells were immersed in the RNase H reaction buffer (5 mm Tris-HCl, 75 mm KCl, 3 mm MgCl 2, 1 mm DTT, ph 8.3) containing 2 units of RNase H (M297, New England Biolabs) at room temperature for 2 h. The samples were rinsed with PBS and examined with AFM. S4
a 6 3-3 b 3 2 1 Force (pn) Count mir-124 mir-134 mir-486 mir-124 mir-134 mir-486 2 4 2 4 2 4 Z-height (nm) n = 1,246 n = 1,3 n = 1,26 23 ± 8 2 ± 6 19 ± 7 c 2 4 6 2 4 6 2 4 6 Force (pn) 3 3.7 ± 1.2 3.9 ± 1.4 3.7 ± 1.2 Count 2 1 5 1 15 5 1 15 5 1 15 Distance (nm) Figure S1. Specific unbinding events for adhesion between HBD and the mirna/dna hybrid. (a) Force-distance curves obtained using three different mirnas. Thirty curves were superimposed for each mirna. (b) Adhesion force histograms. (c) Tip-sample distance histograms for unbinding events. (b, c) n indicates the number of curves analyzed to generate the histograms. The most probable values are given as the means ± s.d. of the Gaussian fit. S5
a - / DNA probe b DNA / DNA c RNA / RNA probe d mir-134 e mir-124 f mir-486 d g mir-134 RNase H 2 4 6 8 1 (%) Figure S2. Recognition of mirna/dna hybrids. To confirm the specificity of the observed unbinding events, we examined ssdna, dsdna, and dsrna spots. The probability of observing specific adhesion force-distance curves on each pixel is represented in grey scale in the force maps. When we scanned a region (2 2, 1. 1. µm 2 ) on each spot by force mapping, pixels showing false-positive unbinding events were rare, and the probability of events was low (a, b, c). However, unbinding events were observed across the whole map when we scanned successively the probe DNA spot capturing complementary mirnas with the same tip (d, e, f). Additionally, the positive signals were dramatically reduced when we re-examined the mir-134- capturing DNA spots after RNase H treatment (2 units, 2 h) (g). Therefore, force measurement confirmed that the binding of HBD and the duplex formed between mirna and DNA is highly specific. (a) Without target on the mir-134 complementary DNA spot. (b f) Target nucleic acids (1 µm) were captured (b, d f) on the complementary DNA spot or (c) on the complementary RNA spot. (b) The DNA homologue of mir-134; (c, d) mir-134; (e) mir-124; and (f) mir-486. (g) mir-134 (1 µm) was captured on a complementary DNA spot, and force maps were acquired before and after the treatment of RNase H using the same AFM tip. S6
a b GSH GST HBD c Hydrodynamic radius 2 4 6 8 1 (%) d e f g Figure S3. Observation of a cluster of positive pixels due to the hydrodynamic distance of HBD and the mirna/dna hybrid. (a) Scheme of Brownian motion of the surface-conjugated HBD and mirna/dna hybrid and their binding at various positions on a dendron-coated glass slide. (b, d, f) High-resolution force maps showing an isolated cluster (pixel size 4. nm). The maps were acquired (b) on the probe DNA spot and (d, f) on the fixed cells. (c, e, g) Each cluster in (b, d, f) was fitted with an ellipse (yellow circle) to calculate its radius. Scale bar, 2 nm. S7
mir-134, 1 fm mir-124, 1 pm mir-134, 1 fm + mir-124, 1 pm (%) 2 4 6 8 1 Figure S4. Selective detection of mir-134. A solution containing 1 fm mir-134, 1 pm mir- 124, or a mixture of two mirnas was incubated on the mir-134 complementary DNA spot. Clusters of positive pixels (yellow circles) were observed only when mir-134s were captured, irrespective of mir-124. The probability of observing a specific unbinding event on each pixel is indicated in grey scale in the force maps (3 3 pixels, 24 24 nm 2 ). S8
a mir-134 pre-mir-134 mir-134 b mir-134, 1 fm pre-mir-134, 1 pm 2 4 6 8 1 (%) c pre-mir-134 (reverse configuration) d pre-mir-134 missing 6-nt at the 5 end Figure S5. Sequential examination of the spots capturing mir-134 and pre-mir-134. The probability of observing a specific unbinding event on each pixel is indicated in grey scale in the force maps (3 3 pixels, 24 24 nm 2 ). Yellow circles represent observed clusters. Upon S9
sequential examination, the HBD tip recognized DNA/miR-134 hybrids but not DNA/RNA with long tails. (a) pre-mir-134; (b) A 1 pm pre-mir-134 solution was incubated on a probe DNA spot to ensure hybridization, whereas 1 fm mir-134 was incubated on the other spot; (c) premir-134 in reverse configuration; and (d) pre-mir-134 missing 6 nt at the 5 end. S1
3-nt overhang at the 5 end 6-nt overhang at the 5 end (%) 2 4 6 8 1 Figure S6. Recognition of mir-134 analogues with a 3-nt or 6-nt overhang. mir-134 with an additional 3- or 6-nt overhang at the 5 end was hybridized to the mir-134 complementary DNA spot. The short overhang up to 6-nt did not interfere the binding of HBD to the mir-134/dna hybrid, and clusters were detected on the force maps. The probability of observing a specific unbinding event on each pixel is indicated in grey scale in the force maps (3 3 pixels, 24 24 nm 2 ). S11
1 am mir-134 hybridized on a 2.9-µm diameter spot 5 am mir-134 hybridized on a 6.4-µm diameter spot 25 am mir-134 hybridized on a 4.9-µm diameter spot 5 am mir-134 hybridized on a 7.-µm diameter spot 25 am mir-134 hybridized on a 4.9-µm diameter spot 1 am mir-134 hybridized on a 5.3-µm diameter spot 1 am mir-134 hybridized on a 7.2-µm diameter spot 2 4 6 8 1 (%) Figure S7. Detection of synthetic mir-134s on probe spots of few microns. We employed an AFM-based fluidic tool to produce DNA spots of a few microns as DNA spots smaller than 1 µm are difficult to produce reliably using a conventional microarrayer. 2 Using the nano-contact tool, the capture DNA solution was delivered to the surface of the glass slides through a 3-nm aperture of a tip connected to a sample solution reservoir. By controlling the contact time and the applied pressure, probe DNA spots of.5 µm to 2 µm in diameter were produced. After the hybridization of mir-134s (1 1 am, 4 µl) to a probe spot, three force maps were acquired on the arbitrary regions of the spot (3 3 pixels, 3 3 nm 2 ). The probability of observing specific unbinding events on each pixel is indicated in grey scale in the force maps. Yellow circles represent observed clusters. S12
a 36 3 b Count 8 6 4 n = 3,243 23 ± 7 24 2 Force (pn) 18 12 c 5 2 4 6 8 Force (pn) 6 Count 4 3 2 1 26 ± -6 1 2 Z-height (nm) 2 4 6 8 1 Distance (nm) d 36 e 2 n = 911 3 Count 15 1 23 ± 7 24 5 Force (pn) 18 12 f 2 2 4 6 8 Force (pn) 6 Count 15 1 5 24 ± 9-6 1 2 Z-height (nm) 2 4 6 8 1 Distance (nm) Figure S8. Unbinding events for adhesion between HBD and mir-134/dna hybrid on fixed cells. (a c) Primary hippocampal neurons (DIV7). (d f) N2a cells. (a, d) Specific force-distance S13
curves that were observed on fixed cells. The baseline of each curve was displaced by 6 pn for clarity. (b, e) Adhesion force histograms. (c, f) Tip-sample distance histograms for unbinding events. (b, c, e, f) n indicates the number of curves analyzed to generate the histograms. The most probable values are given as the means ± s.d. of the Gaussian fit. S14
a MAP2A, green c force map before the hybridization of probe DNA position 1 position 2 position 3 b AFM height image d force map after the hybridization of probe DNA position 1 position 2 position 3 1 2 3 e AFM height image f force map after the hybridization of probe RNA position 1 position 2 position 2 3 1 g AFM height image h force map after the hybridization of scrambled DNA position 1 position 2 position 1 2 3 1.8 (µm) S15
Figure S9. Control experiments to verify the specificity of the observed clusters. (a, b) The boxed region in fluorescence image of fixed neurons (DIV7) was imaged with AFM. (c, d) Numbered positions in the AFM image (b) were scanned by adhesion force mapping before and after the hybridization of mir-134 complementary DNA with the same HBD-tip. (e, f) mir-134 complementary RNA was hybridized, and the numbered positions in the AFM image (e) were examined. (g, h) A solution containing scrambled DNA (22-nt) for mir-134 was incubated before measurement, and the numbered positions in the AFM image (g) were examined. (c, d, f, h) Specific unbinding events were observed more than once out of five measurements at sites indicated by yellow pixels, and clusters are marked by red circles (1 1 pixels, 1. 1. µm 2 ). Scale bar, 5 µm in (a) and 2 µm in (b, e, g). S16
a MAP2 c-fos MAP2 / c-fos b Figure S1. c-fos expression by KCl treatment. Fluorescence images of primary hippocampal neurons (DIV7). MAP2; green, c-fos; red. (a) Unstimulated. (b) Stimulated with 4 mm KCl for 2 h. S17
a bright field AFM height image b AFM height image 3. 1 2 3 1 2 3 (µm) without probe DNA after the hybridization of probe DNA after the incubation of scrambled DNA Force map position 1 position 2 position 3 Figure S11. Detection of mir-134 on fixed N2a cells. Fixed N2a cells were imaged with AFM (scale bar, 2 µm), and the numbered positions in the AFM height images were examined by adhesion force mapping (5 5, 5 5 nm 2 ): (a) before and after the hybridization of probe DNA, and (b) after the incubation of scrambled DNA. The pixels where specific curves were observed with a probability of larger than 2% are colored in yellow. Clusters are indicated by red circles. S18
Name Sequence (5 3 ) Length mir-134 UGU GAC UGG UUG ACC AGA GGG G 22 mir-124 UAA GGC ACG CGG UGA AUG CC 2 mir-486 UCC UGU ACU GAG CUG CCC CGA G 22 DNA homolog of mir-134 TGT GAC TGG TTG ACC AGA GGG G 22 mir-134 with 3-nt appendix at the 5 end GUG UGU GAC UGG UUG ACC AGA GGG G 25 mir-134 with 6-nt appendix at the 5 end pre-mir-134 AGG GUG UGU GAC UGG UUG ACC AGA GGG G 28 AGG GUG UGU GAC UGG UUG ACC AGA GGG GCG UGC ACU CUG UUC ACC CUG UGG GCC ACC UAG UCA CCA ACC CU 71 pre-mir-134 missing 6-nt at the 5 end UGU GAC UGG UUG ACC AGA GGG GCG UGC ACU CUG UUC ACC CUG UGG GCC ACC UAG UCA CCA ACC CU 65 probe DNA for mir-134 (5 -amine) NH 2 -CCC CTC TGG TCA ACC AGT CAC A-Cy3 22 probe DNA for mir-124 (5 -amine) NH 2 -GGC ATT CAC CGC GTG CCT TA-Cy3 2 probe DNA for mir-486 (5 -amine) NH 2 -CTC GGG GCA GCT CAG TAC AGG A-Cy3 22 probe DNA for mir-134 (3 -amine) Cy3-CCC CTC TGG TCA ACC AGT CAC A-NH 2 22 probe RNA for mir-134 (5 -amine) NH 2 -CCC CUC UGG UCA ACC AGU CAC A-Cy3 22 probe DNA for mir-134 CCC CTC TGG TCA ACC AGT CAC A 22 probe RNA for mir-134 CCC CUC UGG UCA ACC AGU CAC A 22 scrambled DNA for mir-134 GGG ATA AAG ATA CCG CTA GTC T 22 Table S1. Sequences of the oligonucleotides used in the experiments. The sequence of mir- 134 within pre-mir-134 and its analogs is colored in blue. S19
Cell Diameter of spot (µm) Number of clusters in a map Mean number of clusters in a map Number of captured mir- 134 on a spot Corrected number considering the capture efficiency Number of mir-134 in a cell Unstimulated 1 5.3 2, 3, 1 2. 176 226 4.7 1, 4, 1.7 116 148 374 Unstimulated 2 5.7, 1,.3 34 44 6.7, 1, 3 1.3 188 241 285 Unstimulated 3 5.2 1, 1,.7 57 73 5.3, 3, 3 2. 176 226 299 KClstimulated 1 7.5 3, 2, 2 2.3 412 529 7.9, 2, 3 1.7 327 419 948 KClstimulated 2 7. 5, 6, 4 5. 77 987 7.9 2, 4, 1 2.3 457 587 1574 KClstimulated 3 7.5 4,, 4 2.7 471 64 8.4 3,, 1 1.3 296 379 983 Table S2. The number of mir-134 in a single cell quantified by AFM force mapping. We isolated total RNA directly from individual neurons using micropipette aspiration and a commercial kit, and counted the number of mir-134s in a single neuron by AFM. To reduce the batch variance effect, we divided the RNA solution from a single neuron into two aliquots and analyzed them on separate spots. Three maps (5 5 pixels, 5 5 nm 2 ) were acquired on each spot, and the mean number of clusters in the three maps was used to calculate the number of captured mir-134 on an entire spot. The numbers of mir-134 on two spots were corrected for S2
capture efficiency (78%) and were added to calculate the amount of mir-134 in a single cell. The coefficient of variation (CV) of the calculated numbers from two spots was approximately 2%, although a higher CV was obtained in two out of six cases (three sets of control cells and three sets of the stimulated cells). It is therefore preferable to examine several sections within a spot and to duplicate the analysis using independent spots. The variance is expected to decrease if a larger area of the spot is scanned or if the homogeneity within the spot is enhanced. REFERENCES (1) Stamatakis, A. M.; Jennings, J. H.; Ung, R. L.; Blair, G. A.; Weinberg, R. J.; Neve, R. L.; Boyce, F.; Mattis, J.; Ramakrishnan, C.; Deisseroth, K.; Stuber, G. D. Neuron 213, 8, 139. (2) Wingren, C.; Borrebaeck, C. A. Drug Discov. Today 27, 12, 813. S21