Supporting Information
A small molecule inhibitor of Pot1 binding to telomeric DNA Sarah E. Altschuler, Johnny E. Croy, and Deborah S. Wuttke
Signal-to-Background
20
15
10
5
0 0.1
1
10
100
1000
[GGTTAC] (nM)
Figure S1. FRET-based competition of unlabeled 6mer against biotind(AAGGTTAC) (5 nM) pre-bound to Pot1pN (10 nM) at 25 °C. Signal-tobackground is plotted as a function of increasing 6mer concentration. Half maximal signal-to-background was achieved at ~25 nM unlabeled 6mer, which is comparable to the measured Pot1pN/6mer KD under these conditions (76).
100
A. 75
Pot1pN KD (nM)
Pot1pN KD (nM)
100
50
25 0
0
5
10
15
% DMSO (v/v)
20
B.
5% DMSO 10% DMSO 20% DMSO
75
50
25
0
5
10
15
20
25
Time DMSO Incubation (h)
Figure S2. The Pot1pN/d(GGTTAC) complex is stable in up to 20% DMSO for at least 24 hours. (A) KD was determined in various DMSO concentrations at ambient temperature using a filter binding assay and plotted as Pot1pN KD (nM) versus % DMSO (v/v). (B) Pot1pN was incubated with the indicated concentrations (v/v) of DMSO for 4, 6, 8 and 24 hours at ambient temperature and subsequently bound to d(GGTTAC). KD is plotted as function of incubation time for each concentration of DMSO.
[M-Na]- = 673.1 Da 13C
ppm
1H
ppm
1H
ppm
Figure S3. 1D 1H and 1H-13C HMBC NMR spectra for Congo red (ST012888). All peaks are within the aromatic and amide regions. Integration of peaks from the 1D spectrum (left) is consistent with CR structure. ESI mass spectrometry revealed a mass of 673.1 Da for the [M-Na] species, consistent with the known mass of 696.7 Da for the CR sodium salt. 1H-13C HMBC NMR spectrum allowed for assignment of small molecule peaks as CR.
Time (min) Time (min)
Power (µcal/sec)
0.10 0.1
00
10
20 20
30
40 40
50
60 60
0.00 0
"#$%&'(#
-0.10 -0.1
!
-0.20 -0.2 -0.30 -0.3 -0.40 -0.4 -0.50 -0.5 0.00 0 -4.00 -4.0 -8.00 -8.0
!
)#$%! *+% ! +.! /01(#2$02
Heat ,-(kcal/mol)
70
-12.00 -12.0
KD = 620 ± 2 nM
-16.00 -16.0
0 0.0
0.5 0.5
1.0 1.0
1.5 1.5
Molar Ratio
2.0 2.0
2.5 3.0 2.5 3.0
Molar Ratio (Pot1pN/CR) Figure S4. Reverse titration of CR and Pot1pN give same KD as forward titration. Baseline-corrected raw data is shown in the top panel, and the integrated heat isotherm is shown in the bottom panel. Twenty-one injections of 272 µM Pot1pN were titrated in 22.5 µM CR at 20 °C. The first injection was 0.2 µL, and all subsequent injections were 1.95 µL.
100
% Mass
80 60 40 20 0
1
10
100
Radius (nm) Figure S5. Addition of a 5-fold excess of 6mer resolves the majority of the CRmediated trimerization. DLS reveals > 80% of the sample mass has a calculated MW of 24.4 kDa, consistent with the size of momomeric Pot1pN. The remaining species appear to be a mixture of dimeric and trimeric Pot1pN complexes. DLS was performed on a DynaPro system (Wyatt Technology Corporation) operating at a wavelength of 633 nm with scattered light detected at 90°. 12 µL samples consisted of 300 µM Pot1pN, 300 µM CR, and 1.5 mM 6mer. Samples were spun down at maximum speed in a microcentrifuge prior to measurement in quartz cuvettes. Data were analyzed using DynaPro Dynamics V 6.3.40 (Wyatt Technology Corporation) to calculate hydrodynamic radius and molecular weight.
