Supplementary Information
Cell-Surface receptor control that depends on the size of a synthetic
equilateral-triangular RNA-Protein complex
Yoshihiko Fujita1, Rie Furushima2, Hirohisa Ohno1, Fumihiko Sagawa1, Tan Inoue1*
1
Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-
cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan.
2
Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka,
Suita, Osaka 565-0871. Japan
*
Tel: +81-75-753-3995; Fax: +81-75-753-3996, E-mail: tan@kuchem.kyoto-u.ac.jp
Contents:
1. Supplementary Results
2. Supplementary Methods
1
Supplementary Results:
Supplementary Figure 1
Characterization of the Tri-RNPs (Tri-RNA-L7Ae complexes). a, RNA EMSA analysis using
the varying concentrations of L7Ae with 100 nM of the indicated Tri-RNAs. The open, gray and
black arrowheads indicate free RNA, RNP with one (or two) L7Ae and RNP with three L7Ae,
respectively. b, Illustration and HS-AFM images of Tri-RNP26m1 and Tri-RNP26m2. The two
point mutations were introduced into the boxC/D motif of Tri-RNA26 to generate the TriRNA26m1 and 2, which fail to interact with one and two L7Ae, respectively. The rectangles in
the background have the width of about 5 nm. The color representation is identical as in Fig. 1. A
scale bar represents 20 nm. c, The images of the designed Tri-RNPs in HS-AFM. A scale bar
represents 50 nm.
Supplementary Figure 2
Characterization of the binding activities of the L7Ae-G1x2 fusion protein. a, RNA EMSA
analysis using the indicated amounts of L7Ae-G1x2 and 100 nM of Tri-RNA48. b, L7Ae and
L7Ae-G1x2 were analyzed on the SDS PAGE gels. One was stained with CBB (left) and the
other was used for the western blotting (right). Each protein was probed with HRP-conjugated
mouse IgG. DC and XP indicate Dual Color Standard marker and MagicMark XP Western
Protein Standard, respectively. c, Identical to Fig. 2-b except that Tri-RNA26 derivatives were
used. A scale bar represents 200 m.
Supplementary Figure 3
Characterization of the Tri-RNA-L7Ae-Gal1 complexes. a, RNA EMSA analysis using the
indicated amounts of L7Ae-Gal1 and 100 nM of Tri-RNA48. b, Bright images of the Jurkat
cells after 6 hours of incubation with the 600 nM of Tri-RNPs (Tri-RNA-L7Ae-Gal1
complexes). A scale bar represents 200 m. c, The Jurkat cells in OptiMEM supplemented with
PI were incubated with the indicated concentrations of the Tri-RNP15 and 92, followed by wash.
The PI fluorescent intensities were quantified by the fluorescent microscopy as in Fig.3-d and
normalized to that of the Tri-RNP15 at 300 nM. The error bars represent the standard deviation
obtained from three independent experiments. d, The Jurkat cells were incubated for 6 hours
with the indicated concentrations of the samples. After stained with the annexin-V conjugated
with Pacific Blue, the dead cells were counted by the flowcytometry and graphed as in Fig.3-f.
The error bars represent the standard deviation obtained from three independent experiments.
Supplementary Figure 4
Promotion and inhibition of apoptosis with Tri-RNP15-L7Ae-Gal1 and lactose, respectively.
a, The Jurkat cells were first stained with the Hoechst 33342, washed and incubated for 7.5 hours
with the indicated concentrations of the RNP complexes to show the nuclear shrinkage. The
nuclei were observed by the fluorescent microscopy and the nuclei of the 49 cells were
represented for the effect of each sample treatment. b, The nuclei shown in the panel a are
evaluated as dead or alive based on the size, circularity, fluorescent intensity and DNA
fragmentation status calculated by the image analysis software. The percentages of the apoptotic
cells were normalized to that of the non-treated control group (OptiMEM). The error bars
represent the standard deviation obtained from two or more independent experiments. c, The
Jurkat cells were incubated with Tri-RNP15 in the presence or absence of 30 mM of lactose.
After stained with the annexin-V conjugated with Pacific Blue, dead cells were counted by the
flowcytometry and graphed as in Fig. 3-f. The error bars represent the standard deviation
obtained from three independent experiments.
Supplementary Methods
Calculation of dissociation constants from EMSA.
The dissociation constants between RNA and proteins were estimated as follows. Assuming that
the dissociation constants of all three box C/Ds in RNA were equivalent and independent from
each other, the dissociation constants can be expressed as the following equation.
Kd = [boxCD][L7]/[boxCD-L7]
= [boxCD]([L7]t – [boxCD-L7])/[boxCD-L7]
[boxCD], [L7], [boxCD-L7] and [L7]t indicate the concentrations of the box C/D, L7Ae, box
C/D occupied by L7Ae, and total L7Ae in the reaction system, respectively. The concentration of
the box C/D in the system and the box C/D occupied by L7Ae were calculated as follows.
[boxCD] = (P0 x 3 + P1 x 2 + P2 x 1)/(P0 + P1 + P2 + P3) x [RNA]t
[boxCD-L7] = (P1 + P2 x 2 + P3 x 3)/(P0 + P1 + P2 + P3) x [RNA]t
P0, P1, P2 or P3 indicate that the intensities of the RNA bands occupied by 0, 1, 2, or 3 L7Ae(s),
respectively. [RNA]t indicates that the concentration of the total RNA in the reaction system.
The intensities were measured by ImageJ software31.
Analysis of apoptotic cells by RS-100 imager.
Jurkat cells were stained in culture medium supplemented with 10 g/mL of Hoechst 33342 for
30 min followed by wash twice. RNP solutions were prepared in the same way described for the
flow cytometry analysis. The stained cells were dispensed in the RNP solutions in a 96 well plate
and shaken for 10 sec on a microshaker. The cell suspension with RNP was incubated for about
30 min until the cells settle down to the bottom. The images of the nucleus of the Jurkat cells was
captured on RS-100 by 365 nm LED and a 420 nm low bandpass filter every hour. The apoptotic
cells were detected with the image analyzing software of RS-100 imager. The conditions for the
dead and apoptotic cells are as follows:
a OR (b AND c AND d AND (e OR g OR (f AND h)))
(a) An area of nucleus is smaller than the arbitrary value. (b) Circular factor is higher than the
arbitrary value. (c) The mean intensity of fragmented nuclei is higher than the arbitrary value. (d)
The area of fragmented nuclei is in the arbitrary range. (e) The occupancy of the fragmented
nuclei is higher than the arbitrary value. (f) The occupancy of the fragmented nuclei is in the
arbitrary range. (g) The number of the fragmented nuclei is equal to or more than two. (h) The
mean intensity of a whole nucleus is higher than the arbitrary value.