1

Supporting Information

An RNA aptamer with potent affinity for a toxic dimer of amyloid b42 has potential utility for histochemical studies of Alzheimer’s disease*

Kazuma Murakami,1,* Yayoi Obata,1 Asa Sekikawa,1 Haruka Ueda,1 Naotaka Izuo,2,#

Tatsuya Awano,3 Keiji Takabe,3 Takahiko Shimizu,2,§ and Kazuhiro Irie1,†

From 1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan; 2Department of Endocrinology, Hematology and Gerontology, Graduate School of

Medicine, Chiba University, Chiba, 260-8670, Japan; 3Division of Forest and Biomaterials Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

*Running title: RNA aptamers targeting Ab42 toxic dimer

#Department of Pharmaceutical Therapy and Neuropharmacology, Faculty of Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan §Present address: Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, 474-8511, Japan *To whom correspondence should be addressed: Kazuma Murakami, Ph.D., Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: +81-75-753-6282, Fax: +81-75-753-6284, E-mail: alzkazu@kais.kyoto-u.ac.jp (ORCiD: 0000-0003-3152-1784) †To whom correspondence should be addressed: Kazuhiro Irie, Ph.D., Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: +81-75-753-6281, Fax: +81-75-753-6284, E-mail: irie@kais.kyoto-u.ac.jp (ORCiD: 0000-0001-7109-8568) [Contents] Figure S1~S8 Table S1~S2

1

Figure S1. TEM to evaluate the preparation of PFs. The aggregates were produced from (A) biotin-E22P-V40DAP-Aβ42 dimer or (B) wild-type Aβ40 (25 μM) after incubation for 48 hr at 37°C. Arrowheads indicate PFs or high-order oligomers with curvilinear structures. Scale bar = 100 nm.

A

B

3

Figure S2. Overview of SELEX adopted in this study. RNA aptamers that target PFs of Aβ42 were selected using the method of membrane filter whose cut off of molecular weight is 50 kDa.

4

Figure S3. Alignment search, classification, and local supermotif analysis of E22P-AbD4, -AbD31 and -AbD43. A, Alignment analysis of random sequence by Clustal Omega. Asterisks indicate positions which have a fully conserved residue. B, Classification and local supermotif analysis by Multiple EM for Motif Elucidation (MEME). The height of a letter indicates its relative frequency at the given position (x-axis) in the motif. E22P-AbD43 unlikely has a specific common motif with E22P-AbD4 and -AbD31.

5

Figure S4. Characterization of E22P-AbD4, -AbD31 and -AbD43 binding to Aβ. A, BLI sensorgram and curve fitting of the indicated aptamer (2:1 heterogeneous ligand binding model) to PFs as an analyte with the concentration of Aβ equal to: blue: 200; orange: 400; green: 800; and black: 1600 nM. B, BLI sensorgram and curve fitting of E22P-AbD43 (2:1 heterogeneous ligand binding model) as a ligand to wild-type Aβ40, PFs prepared from wild-type Aβ40, wild-type Aβ40 fibril as the analyte with the concentration of Aβ equal to: blue: 200; orange: 400; green: 800; and black: 1600 nM. KD1 and KD2 values are indicated. N.D. = not determined.

6

Figure S5. Characterization of E22P-AbD4(Ran29), -AbD31(Ran29) and -AbD43(Ran29) binding to PFs. BLI sensorgram and curve fitting of the indicated aptamer (1:1 binding model) as a ligand to PFs as the analyte with the concentration of Aβ shown as blue: 200; orange: 400; green: 800; black: 1600 nM. KD values are indicated. The upward trends at the beginning of the dissociation could be due to bulk effect, which generally originates from the difference in liquid composition between the solution (e.g. ammonium hydroxide solution) for analyte preparation and the running buffer.

-0.01

0.01

0.03

0.05

0.07

0.09

0 100 200 300-0.01

0.01

0.03

0.05

0.07

0.09

0 100 200 300-0.01

0.01

0.03

0.05

0.07

0.09

0 100 200 300Time (sec)

KD = 350 nM

Shift

(nm

)

Time (sec)Time (sec)

E22P-AbD4(Ran29) E22P-AbD31(Ran29) E22P-AbD43(Ran29)

KD = 170 nMKD = 200 nM

Figure S5

0.08

0.06

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0

0.08

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Figure S6. Analysis of secondary structure of RNA aptamer targeting PFs by ATR-FTIR. FTIR spectrum of E22P-AbD43 was obtained by ATR method using anti-Aβ aptamer forming G-quadruplex as a reference (T-SO508) (14,27). A droplet (5~10 µg) of aptamer solution with PBS containing 1 mM EDTA was loaded and then air-dried on an ATR unit.

Figure S4

1800 1700 1600 1500

0

0.1

0.2

0.3

Wavenumber (cm-1)

Abso

rban

ce

E22P-AbD43

T-SO508

1649

8

Figure S7. Fluorescence polarization of E22P-AbD43 binding to Aβ. Fluorescence polarization curves showing binding profiles of E22P-AbD43 with Aβ42 toxic dimer, wild-type Aβ42, E22P-Aβ42, and wild-type Aβ42 fibril. KD values are indicated. Data are expressed as the mean ± s.d. (n = 3).

Figure S7

WT-Aβ42 (KD = 160 nM)E22P-Aβ42 (KD = 120 nM)

Aβ42 toxic dimer (KD = 54 nM)

WT-Aβ42 fibril (KD = not determined)140

1

160

180

200

220

Aβ (nM)

Fluo

resc

ence

pol

ariza

tion

(mP)

12010 100-9 -8 -7

120

140

160

180

200

220

Aβ [log(M)]

Fluo

resc

ence

pol

ariz

atio

n (m

P)

Aβ42 toxic dimerWT-Aβ42

WT-Aβ42 fibril

X-6.500000-6.800000-7.100000-7.400000-7.700000-8.000000-8.300000-8.600000-8.900000-9.200000

Aβ42 toxic dimerY1204196188160154147145145146145

Y2194193189163152146145145145144

Y3198196175160151144137141142137

WT-Aβ42Y1188172157155148146144145144153

Y2185174158153148146142145145153

Y3182171160153146147144145144152

E22P-Aβ42Y1169170155150148147149148148149

Y2168163152153150148148148148150

Y3176165157156152148150150150150

WT-Aβ42 fibrilY1153152155155151147150149151151

Y2152153155155152147149149150151

Y3153154154155152145148146146147

Sigmoidal, 4PL, X is log(concentration)Best-fit values

TopBottomLogIC50HillSlopeIC50Span

Std. ErrorTopBottomLogIC50HillSlopeSpan

95% Confidence IntervalsTopBottomLogIC50HillSlopeIC50Span

Goodness of FitDegrees of FreedomR squareAbsolute Sum of SquaresSy.x

Number of pointsAnalyzed

Aβ42 toxic dimer

200.6143.3-7.2682.0535.401e-00857.24

2.5231.0360.035620.29362.923

195.4 to 205.8141.2 to 145.5-7.341 to -7.1941.449 to 2.6564.563e-008 to 6.393e-00851.23 to 63.25

260.9758348.43.660

30

WT-Aβ42

198.5146.2-6.7951.5581.602e-00752.25

10.830.83090.13690.360511.17

176.2 to 220.7144.5 to 147.9-7.077 to -6.5140.8168 to 2.2998.377e-008 to 3.063e-00729.29 to 75.21

260.9560226.32.950

30

E22P-Aβ42

174.2149.0-6.9152.1261.215e-00725.28

3.5640.58780.083240.58143.753

166.9 to 181.6147.8 to 150.2-7.087 to -6.7440.9302 to 3.3218.194e-008 to 1.802e-00717.56 to 32.99

260.9251141.22.330

30

WT-Aβ42 fibrilAmbiguous

153.8~ 148.4~ -7.701~ 194.0~ 1.991e-008~ 5.433

0.4802~ 9.031e+006~ 5.903e+014~ 1.246e+020~ 9.031e+006

152.8 to 154.8(Very wide)(Very wide)(Very wide)(Very wide)(Very wide)

260.734371.931.663

30

E22P-Aβ42

9

Figure S8. Comparison of the numbers of diffuse aggregates and senile plaques stained in Figure 5 in the main text. Three parts in (A) E22P-AbD43 and (B) 82E1 are used for counting of number of diffuse aggregates and senile plaques, respectively, and (C) summary of counting. In (A, B), scale bars show 500 µm.

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Table S1. Calculated kinetic parameters for kon, koff, KD of full-lengtha or randomb E22P-AbD4, -AbD31, and -AbD43 (ligand) with Aβ (analyte) using the curve fitting used in this study. aThese parameters are derived from the curve fitting of data with a 2:1 heterogeneous ligand binding model. bThese parameters are derived from the curve fitting of data with a 1:1 binding model. cThe values in the parentheses indicate standard error. dThe values in the first line indicate kon1, koff1, and KD1. eThe values in the second line indicate kon2, koff2, and KD2. fNot determined due to no significant binding. gThe percent contributions from KD1 and KD2 in the case of a 2:1 heterogeneous ligand model. N.D. = not determined N.A. = not available

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Table S2. Calculated kinetic parameters for kon, koff, KD of full-lengtha or randomb E22P-AbD4, -AbD31, and -AbD43 (ligand) with Aβ (analyte) using the curve fitting un-used in Figures. aThese parameters are derived from the curve fitting of data with a 2:1 heterogeneous ligand binding model. bThese parameters are derived from the curve fitting of data with a 1:1 binding model. cThe values in the parentheses indicate standard error. dThe values in the first line indicate kon1, koff1, and KD1. eThe values in the second line indicate kon2, koff2, and KD2. fNot determined due to no significant binding. gThe percent contributions from KD1 and KD2 in the case of a 2:1 heterogeneous ligand model. N.D. = not determined N.A. = not available