Analytical and Bioanalytical Chemistry

ELECTRONIC SUPPLEMENTARY MATERIAL

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

CHAYAN K. NANDI, PARTHA P. PARUI, THORSTEN L. SCHMIDT, ALEXANDER HECKEL, BERNHARD

BRUTSCHY

From a fluorescence correlation function one can get many important quantities such as the number of particles as well as the concentration and the diffusion times of a labeled molecule. This information drives many researchers for the binding studies of biomolecules. FCS is a very important experimental method due to its high sensitivity, accurate and convenient analytical properties by means of confocal optics. But these parameters are affected by photochemical processes as well as experimental artifacts. These artifacts mainly originate from the utilized excitation power. The deviation from linearity of the fluorescence intensity with increasing excitation power causes the distortion of the fluorescence correlation function. So, one should be very careful when performing a FCS experiment. A smaller excitation power reduces the chances of artifacts and ensures an accurate measurement of the absolute value of these parameters. At the same time, measurements at a fixed excitation power can be used for the quantitative determination of the relative values of concentrations and diffusion times.

A. Excitation intensity dependent FCS experiment

We have performed FCS binding studies of hairpin polyamides to DNA using fluorescence correlation spectroscopy (FCS) in the auto- and cross-correlation mode in a laser power range where the fluorescence intensity as well the brightnesses (η) for both the RhG labeled free polyamides as well as of the DNA-bound polyamides shows a linear dependence with the excitation laser power. Similarly the number of particles as well as the diffusion times do not

1 deviate from constant value for the free as well as the bound molecules. The laser power used for our experiment is less than 200 μW. Figures S1a and S1b show the change in fluorescence intensity per molecule (kcps/molecule) with increasing excitation power for the

RhG labeled free polyamide (Pb) and RhG labelled DNA-bound polyamide (Pb:Db) at 1:1 stoichiometry. Figures S2a and S2b show the change in number of particles with increasing excitation power for the RhG labelled free polyamides (Pb) and RhG labelled DNA-bound polyamide (Pb:Db) at 1:1 stoichiometry. The data shows that the deviation from the linearity occurs only at a laser power above 800 μW. The number of particles and the diffusion time do not change in this region of laser power.

100 ) ) s s e e l l u u c c e e l l

m m 10 / / s s p p c c k k ( (

  (a) (b)

1 1 10 100 1000 10 100 1000 Excitation power (W) Excitation power (W)

Fig S1: (a) The change in fluorescence intensity per molecule with increasing excitation power for the RhG labeled free polyamide (Pb). (b) The change in fluorescence intensity per molecule with increasing excitation power for the RhG labeled DNA-bound polyamide (Pb:Db) at 1:1 stoichiometry.

3,0 5,0

4,5 2,5

4,0 s s e e l 2,0 l c c i i 3,5 t t r r a a P 1,5 P 3,0

f f o o

r r 2,5 e 1,0 e b b m m 2,0 u u N 0,5 N (c) 1,5 (d)

0,0 1,0 10 100 1000 10 100 1000 Excitation Power (W) Excitation power (W)

Fig S2: (a) Change in number of particles with increasing excitation power for the RhG labeled free polyamide (Pb) and (b) change in number of particles with increasing excitation power for the RhG labeled DNA-bound polyamides at 1:1 stoichiometry (Pb:Db).

2 B. Fitting of raw FCS data to one and two component model

We show the fitting of the FCS data to a two component model for the Pb:Db complex and for free polyamide. For the two component fitting curve we have used the diffusion time of the free polyamides (30 μs) and the diffusion time of the Pb:Db complex in saturation as 63 μs. By this procedure we get the fraction of the complex formation with increasing concentration of 1,4

1,3 n o i t a l

e 1,2 r

r o c o t u A 1,1

1,0 10 100 1000 10000  ( s) 1,4

1,3 n o i t a l

e 1,2 r

r o c o t u A 1,1

1,0 10 100 1000 10000  (s)

Fig S3: (a) Fluorescence auto correlation function of Pb:Db complex in one component model, when 50 % of the complex is formed (b) The same curve is fitted to a two component model. Solid line is the auto-correlation function and dotted line is the fitted one.

3 Db when Pb remains constant. Figure S3a shows fluorescence auto correlation function of the

Pb:Db complex when 50 % complex is formed. The curve is fitted two one component model. Figure S3b shows the same curve fitted by the two component model. The two component model fitting curve is well-matched with the auto correlation curve. In Figure S4 the solid line depicts the residual of two component fitting of the auto correlation curve while the dotted line is the residual of one component fitting. Figure S5 shows the residuals of the fitting of the fluorescence auto correlation function of the

Pb:Db complex when 75 % of the complex is formed. Figure S6 shows the residuals of fitting of the fluorescence auto correlation function of RhG labeled free polyamide Pb without the addition of Db to the system.

0,005 s l a

u 0,000

d i s e

R -0,005

-0,010

-0,015

-0,020 10 100 1000 10000 100000  (s)

Fig S4: Residuals of fitting of the fluorescence auto correlation function of Fig S3. The solid line shows the two component fitting of the auto correlation curve and dotted line is one component fitting.

0,01 s l a

u 0,00

d i s e R -0,01

-0,03 10 100 10004 10000 100000  (s) Fig S5: Residuals of fitting of the fluorescence auto correlation function of Pb:Db complex when 75 % of the complex is formed. The solid line shows the residual of two component fitting and the dotted line from one component fitting.

0,005 s l a

u 0,000

e -0,005 R -0,010

-0,015

-0,020

-0,025

-0,030 10 100 1000 10000 100000  (s)

Fig S6: Residuals of fitting of the fluorescence auto correlation function of RhG labeled free polyamide Pb without the addition of the DNA Db to the system. The curve is fitted to the two component model. Solid line shows the two component fitting and the dotted line shows one component fitting.