1

Supporting Information for

DNA-Based Assemblies for Photochemical Upconversion

Saymore Mutsamwira,a Eric W. Ainscough,

a Ashton C. Partridge,

a,b Peter J. Derrick

a,b and

Vyacheslav V. Filichev*a

aInstitute of Fundamental Sciences, Massey University, Private Bag 11-222, Palmerston

North, New Zealand.

b Department of Physics and School of Engineering, The University of Auckland, 20

Symonds Street, Auckland, New Zealand.

2

Table of Contents

1. Materials ............................................................................................................................................. 3

2. Synthesis and Purification of TINA Modified Oligonucleotides .......................................................... 3

3. Stock Solutions .................................................................................................................................... 3

4. DNA annealing procedures ................................................................................................................. 4

5. Instrumentation .................................................................................................................................. 4

5.1 UV-Vis Spectroscopy, DNA melting procedure and Thermal Difference Spectra (TDS) ............... 4

5.2 Circular Dichroism Experiments .................................................................................................... 5

5.3 Fluorescence Spectroscopy ........................................................................................................... 5

5.4 Upconverted fluorescence measurements ................................................................................... 5

6. UV–vis spectroscopy of [Ru(bpy)3]2+/TINA-DNA complexes ............................................................... 7

7. Fluorescence spectroscopy of [Ru(bpy)3]2+/PEPy complexes ........................................................... 10

8. Fluorescence spectroscopy of [Ru(bpy)3]2+/TINA-DNA complexes ................................................... 11

9. K4Fe(CN)6 – [Ru(bpy)3]2+ Fluorescence Quenching Studies ............................................................... 14

10. UV-Vis thermal difference spectra (TDS) ........................................................................................ 16

11. Circular Dichroism (CD) spectra ...................................................................................................... 18

12. ZnTMpyP4/ TINA – DNAs PUC trials ................................................................................................ 19

13. Characterization of modified DNAs. ............................................................................................... 20

13.1 MALDI-TOF spectra ................................................................................................................... 20

13.2. Denaturing Polyacrylamide gel electrophoresis of modified DNAs. ........................................ 24

References ............................................................................................................................................ 26

3

1. Materials

All organic solvents were purchased from Sigma–Aldrich, Fluka and Fisher Scientific.

Unmodified oligonucleotides were purchased from Integrated DNA Technologies. (R)-1-O-

[4-(1-pyrenylethynyl)phenylmethyl]glycerol (PEPy) and [Ru(bpy)3](BF4)2 were synthesized

previously in our laboratory according to published procedures.1, 2 All aqueous solutions

were prepared in ultrapure MilliQ water (18.2 MΩ·cm).

2. Synthesis and Purification of TINA Modified Oligonucleotides

Oligonucleotides were synthesized on a 1.0 μmol, 1000 Å CPG supports with an Mer-Maid 4

automated DNA synthesizer from BioAutomation Corporation using 4,5-dicyanoimidazole in

dry acetonitrile as activator. The synthesis was paused for TINA coupling after the

detritilation step. TINA was hand coupled by adding the dry monomer (10 mg) on top of the

column followed by 200 μl of activator (0.25 M DCI in dry acetonitrile) that was directly

injected into the column under argon after which the synthesis cycle was allowed to

continue. The DNA synthesis was completed in DMT-off mode. The oligonucleotides were

cleaved from the solid support, deprotected, and purified according to a previously

published procedure.3 Purity of TINA-modified oligonucleotides was monitored using

denaturing 20% PAGE (7 M urea) and was shown to be over 90 %. Molecular weights of

oligonucleotide sequences were determined using a Waters Micromass MALDI-TOF in the

positive mode. Oligonucleotides were desalted using C18 zip-tips prior to loading on the

MALDI plate, using anthranilic acid as a matrix and dibasic ammonium citrate as a co-matrix;

see section 13 for representative pictures of denaturing PAGE and mass-spectra.

3. Stock Solutions

[Ru(bpy)3]2+: A 0.5 mM stock solution was prepared by weighing the [Ru(bpy)3](BF4)2 salt

(23.84133 mg) on an ultra-sensitive analytical balance followed by dissolution of the solute

in MilliQ H2O (100 mL). The concentration was confirmed by UV-Vis spectroscopy, using an

extinction coefficient at 452 nm of 14 600 L mol-1 cm-1 corresponding to the MLCT

transition.4

Oligonucleotides: Concentrations of stock solutions of single stranded oligonucleotides were

determined by UV-Vis spectroscopy (see below for details).

4

4. DNA annealing procedures

DNA duplexes were prepared by first suspending both complementary single stranded

oligonucleotides at the same molar concentration in MilliQ H2O. The equimolar

complementary oligonucleotides were mixed in a 1.5 ml eppendorf tube, followed by the

addition of the appropriate annealing buffer. The eppendorf tube was then placed in a

heatblock at 90 °C for 10 minutes, removed from the heat block and allowed to cool to

room temperature.

5. Instrumentation

5.1 UV-Vis Spectroscopy, DNA melting procedure and Thermal Difference Spectra

(TDS)

UV-Vis spectra for [Ru(bpy)3]2+ and oligonucleotides were collected using a Shimadzu UV-

3101PC UV-VIS-NIR-scanning spectrophotometer in 1 mL cuvettes at a pathlength of 1 cm.

UV-Vis spectra were taken at a concentration of 1.0 μM of each oligonucleotide in 10 mM

sodium phosphate buffer (pH 7), 0.1 mM EDTA at low salt concentration (50 mM NaCl) and

high salt concentration (1 M NaCl) at room temperature. The concentrations of [Ru(bpy)3]2+

were 40 or 100 µM. Melting temperature measurements of oligonucleotides were collected

on a CARY 100 Bio UV-Vis spectrophotometer using a 2 × 6 multicell block with a Peltier

temperature controller from 20 to 80 °C at a rate of 1 °C/min. All melting temperatures are

within the uncertainty of ± 0.5 °C as determined by repetitive experiments. Extinction

coefficients for oligonucleotides were calculated using the extinction coefficients of each

nucleotide and TINA at 260 nm. Extinction coefficients of nucleotides and TINA (L mol-1 cm-

1): dA (15400), dG (11700), dT (8800), dC (7300), TINA (22400). Thermal difference spectra

for DNA complexes were determined by subtracting the UV-Vis spectra obtained at 20 °C

from UV-Vis spectra obtained at 90 °C after 30 mins incubation in the appropriate buffer

solutions.

5

5.2 Circular Dichroism Experiments

CD spectra were recorded on an Applied Photophysics Chirascan CD spectrometer (150 W

Xe arc) with a Quantum Northwest TC125 temperature controller using quartz cuvettes

with an optical path length of 1 cm. All measurements were done at 25 °C. An average of

three scans between 210 nm and 700 nm was recorded at 1 nm intervals, 240 nm/min

scanning speed. A baseline correction was applied against the appropriate buffer followed

by data smoothing using the Savitzky-Golay method. Data was recorded in mdeg and

converted to delta epsilon from the formula ∆=(mdeg)/32980 × C × l using software

provided by Applied Photophysics. The [Ru(bpy)3]2+ and DNA-[Ru(bpy)3]2+ samples were

prepared in the same manner and in the same buffer solutions as for UV-Vis measurements.

Concentrations of stock solutions were chosen such that the total dilution of the analyte

solution did not exceed 1% by each addition of [Ru(bpy)3]2+. CD spectra were recorded after

each incremental addition of [Ru(bpy)3]2+, after which the solutions had reached

equilibration (≥20 min).

5.3 Fluorescence Spectroscopy

Fluorescence data were collected using a FluoroMax-4 spectrofluorimeter (Horiba Scientific

Jobin Yvon) or a Perkin Elmer LS-50 Luminescence Spectrometer using 1 cm path length

quartz cuvettes, volume 1 mL. The [Ru(bpy)3]2+ and DNA-[Ru(bpy)3]2+ samples were prepared

in the same manner and in the same buffer solutions as for UV-Vis measurements. The

fluorescence emission of [Ru(bpy)3]2+, the DNAs alone, and the DNA-[Ru(bpy)3]2+ complexes

were measured at a wavelength range of 390-800 nm for TINA excitation (λex = 375 nm), and

of 520-800 nm for [Ru(bpy)3]2+ excitation (λex = 500 nm). The excitation spectra were

recorded at a wavelength range of 200-400 nm (λem TINA: 407 nm), and 200-590 nm (λem

[Ru(bpy)3]2+: 600 nm).

5.4 Upconverted fluorescence measurements

Energy upconversion solutions containing [Ru(bpy)3]2+ (46 µM) and PEPy (4.6 mM) in

dichloromethane (DCM) were prepared from stock solutions of 0.5 mM [Ru(bpy)3]2+ and 5

mM PEPy. Solution of ZnTMpyP4 (5.0 – 200 µM), from a 0.5 mM stock and PEPy (0.01 – 5

6

mM) were prepared in a in 9:1 (DMSO/H2O) solvent mixture. These solutions were

deaerated by purging with argon gas in a sonicator for at least 30 min and were kept under

argon in a silicon septum fitted quartz cuvette throughout the experiments. Aqueous

solutions of TINA-modified DNAs with ZnTMpyP4 or [Ru(bpy)3]2+ were prepared in 10 mM

sodium phosphate buffer (pH 7.0, 0.1 mM EDTA, 50 mM NaCl) and then slowly degassed by

argon purging at a rate of 5 bubbles/ minute for 24 hours using needle (19G×1½’’). Fast

argon purging at higher bubble rates was not sufficient to observe upconversion in the

buffer solutions.

Anti-Stokes fluorescence spectra were measured on all deaerated samples with the

excitation at 500 nm for [Ru(bpy)3]2+ and 565 nm for ZnTMpyP4 passing through a 400 nm

long pass filter prior to incidence on the sample. The incident power dependence of PUC

was measured by systematically varying the excitation power through the use of neutral

density filters.

7

6. UV–vis spectroscopy of [Ru(bpy)3]2+

/TINA-DNA complexes

The absorption spectrum of [Ru(bpy)3]2+ shows peaks in the UV range between 200 – 300

nm which are assigned to ligand-ligand transitions (L-L) while the long wavelength

absorption around 450 nm is assigned to a metal to ligand charge transfer (1MLCT), Figure

3A.5, 6 The MLCT photoluminescence band of [Ru(bpy)3]2+ is centered at 600 nm (Figure 3A).5

PEPy (TINA monomer) exhibits absorption bands between 350 – 400 nm, red-shifted

compared to pyrene due to π-conjugation extension,7, 8 with the monomer fluorescence

maximum at ~420 nm.

Hyperchromic shifts were observed for the MLCT [Ru(bpy)3]2+ band in the presence of TINA-

modified DNA duplexes (Figure 6), in contrast to the observation of hypochromic shifts

when long stranded DNAs of biological origin such as salmon testes DNA (stDNA) are added

to [Ru(bpy)3]2+ due to stacking interactions with bases.9-11 In the presence of TINA-modified

DNAs, TINA affects the normal stacking interactions between the base pairs and [Ru(bpy)3]2+

as a result of [Ru(bpy)3]2+ being drawn more to the lipophilic TINA than to the base pairs.

The increase in the π-π* intraligand absorption band around 286 nm on DNA binding is a

result of an additive effect due to an overlap with the 260 nm band from DNA nucleobases

(Figure SI 1, also see Table SI 1).

Increasing the concentration of NaCl from 50 mM to 1 M generally results in an increase of

the [Ru(bpy)3]2+ visible absorption band (Table SI 1), due to competition with Na+ for the

polyphosphate backbone on DNA, which in turn results in [Ru(bpy)3]2+ being more drifting to

the lipophilic pyrenyl groups. A dramatic increase in the MLCT band with two TINAs

attached, D3 and D4, both in low and high salt concentration solutions means more TINA-

[Ru(bpy)3]2+ attraction occurs as the number of TINAs increase. The largest increase was

seen with D4 in the high salt concentration (H = +36.4 %).However, no bathochromic or

hypsochromic shifts were observed.

The TINA absorption was also perturbed on addition of [Ru(bpy)3]2+. In all duplexes studied

the 373 nm band was masked by the [Ru(bpy)3]2+ background absorbance (Table SI 2). For

D3 both the 373 nm and 396 nm peaks were not detectable. Duplex D2 had slight

bathochromic shifts in the 396 nm peak to 399 nm (low salt) and 398 nm (high salt) and

hypochromic shifts (-64.1 % low salt, -78.5 % high salt). Duplexes D3 and D4 showed

8

hyperchromic shifts (≈ 30 %). The extents of the shifts were higher in the high salt

concentration.

Figure SI 1. UV-Vis absorption spectra of free [Ru(bpy)3]2+ (40 μM), free D2 (1.0 μM), and D2 in the

presence of [Ru(bpy)3]2+. Conditions: 25 °C, pH = 7.0, 10 mM sodium phosphate buffer, 0.1 mM

EDTA, 50 mM NaCl.

Table SI 1 UV-Vis absorption data for [Ru(bpy)3]2+ bound to duplexes.a

Complex MLCT band ,

nm MLCT band, %

∆Ab

L-L band, nm L-L band, % ∆Ab

L[Ru(bpy)3]

2+ 454 - 286 - H[Ru(bpy)3]

2+ 453 - 286 - LD1 + [Ru(bpy)3]2+ 453 +2.1 286 +10.6 HD1 + [Ru(bpy)3]2+ 453 +5.6 285 +12.6 LD2 + [Ru(bpy)3]

2+ 453 +1.2 285 +14.0 HD2+ [Ru(bpy)3]2+ 453 +6.3 286 +14.0 LD3+ [Ru(bpy)3]2+ 453 +22.7 285 +11.0 HD3+ [Ru(bpy)3]

2+ 454 +27.9 286 +8.0 LD4+ [Ru(bpy)3]

2+ 454 +24.1 286 +18.6 HD4+ [Ru(bpy)3]2+ 453 +36.4 286 +18.1

a [Ru(bpy)3]2+ = 40 μM, duplex = 1.0 μM, 10 mM sodium phosphate buffer, 0.1 mM EDTA, pH = 7.0 in the presence of 50 mM (superscript L) and 1.0 M NaCl (superscript H), 25 ˚C. b % ∆A = [(Afree – Abound)/ Afree] × 100. Afree and Abound are the absorbances of free and bound [Ru(bpy)3]2+, respectively.12, 13

9

Table SI 2 UV-Vis absorption parameters for the pyrenyl moiety of TINA on duplexes bound to [Ru(bpy)3]2+. % ∆A was

calculated as in Table 2.

Duplex λmax % ∆A (≈396 nm) % ∆A (≈373 nm) LD2 373;396 - -

HD2 373;396 - -

LD3 371;392 - - HD3 373;396 - - LD4 371 - -

HD4 394 - - LD2 + [Ru(bpy)3]2+ 399 -64.1 *NV HD2+ [Ru(bpy)3]

2+ 398 -78.5 *NV LD3+ [Ru(bpy)3]

2+ 395 +33.1 *NV HD3+ [Ru(bpy)3]2+ *NV *NV *NV LD4+ [Ru(bpy)3]2+ 394 +25.6 *NV HD4+ [Ru(bpy)3]

2+ 399 +30.6 *NV Buffer conditions are the same as in Table SI 1. *NV - there is no peak at 396 nm (no value to report).

10

7. Fluorescence spectroscopy of [Ru(bpy)3]2+

/PEPy complexes

The steady state MLCT fluorescence emission of [Ru(bpy)3]2+ upon 500 nm excitation was

efficiently quenched by PEPy (Figure 3C) and the extent of quenching was quantified by the

Stern-Volmer relation (eqn. 1 SI):

F0/F = 1 + KSV[Q] eqn 1 SI

where F0 and F are the photoluminescence intensities in the absence and presence of

quencher, respectively; [Q] is the molar concentration of quencher. From the slope of the

Stern-Volmer plot of F0/F versus [Q] at 25 ̊C (Figure 3D), the Stern-Volmer quenching

constant value (Ksv) for [Ru(bpy)3]2+ quenching by PEPy was found to be 91.5 M-1, thereby

yielding a bimolecular quenching constant (kq) of 1.31×108 M-1 s-1 calculated from eqn. 2 SI

and using τ0 (700 ns):14

Ksv = τokq eqn 2 SI

where τ0 is the lifetime of [Ru(bpy)3]2+ in the absence of quencher (PEPy).

[Ru(bpy)3]2+ also quenches the steady-state fluorescence of PEPy:

Figure SI 2 (A) Steady state fluorescence quenching of PEPy (0.25 µM) by [Ru(bpy)3]2+ in the quencher concentration range of 0.0 – 30.0 µM in DCM at 25 ˚C, λex = 375 nm, insert picture shows energy transfer from PEPy to [Ru(bpy)3]2+. (B) A Stern-Volmer plot for the quenching of the fluorescence of PEPy (0.25 μM) by [Ru(bpy)3]2+.

11

8. Fluorescence spectroscopy of [Ru(bpy)3]2+

/TINA-DNA complexes

Table SI 3 Fluorescence emission data for [Ru(bpy)3]2+ bound to duplexes, λex = 500 nm.

Complex λmax , nm Fluorescence

enhancement, % L[Ru(bpy)3]2+ 598 - H[Ru(bpy)3]2+ 596 -

LD1 + [Ru(bpy)3]

2+ 596 9 H

D1 + [Ru(bpy)3]2+ 597 4

LD2 + [Ru(bpy)3]2+ 599 17 HD2+ [Ru(bpy)3]

2+ 596 8 LD3+ [Ru(bpy)3]

2+ 597 11 HD3+ [Ru(bpy)3]2+ 600 10 LD4+ [Ru(bpy)3]2+ 599 10 HD4+ [Ru(bpy)3]

2+ 599 8 Conditions are the same as in Table SI 1.

Figure SI 3. Fluorescence quenching of TINA D3 (1.0 µM) by [Ru(bpy)3]2+. Insert picture shows a [Ru(bpy)3]2+ energy transfer from TINA excitation, λex = 375 nm. Conditions: 25 ˚C, pH = 7.0, 10 mM sodium phosphate buffer, 0.1 mM EDTA, 50 mM NaCl.

12

Table SI 4 Fluorescence emission characteristics of TINA and TINA-[Ru(bpy)3]2+ complexes upon excitation of TINA at 375

nm.

Duplex TINA emission (λmax, nm) [Ru(bpy)3]2+

emission (λmax,

nm)

TINA, Iex/Im Monomer Excimer

D2L 407;427 - - - D2

H 407;427 - - -

D2L + [Ru(bpy)3]2+ 408 - 602 - D2H + [Ru(bpy)3]2+ 406 - 599 -

D3L 415;427 498 - 0.77

D3H 415;429 492 - 1.3

D3L + [Ru(bpy)3]2+ 403;415 498 600 0.70 D3H + [Ru(bpy)3]2+ 404 500 593 0.26

D4L 407;427 501 - 0.55

D4H 409;427 496 - 0.48 D4L + [Ru(bpy)3]2+ 407 503 596 0.32 D4

H + [Ru(bpy)3]

2+ 407 500 598 0.18 Buffer and experimental conditions, see Table SI 1. Iex/Im = Iexcimer(500nm)/Imonomer (405 nm)

Figure SI 4. Fluorescence excitation spectra for D3 (1.0 µM) in presence of [Ru(bpy)3]2+ (40 µM) at [Ru(bpy)3]2+ 600 nm emissionR, and TINA 490 nmT emission. Superscript R denotes [Ru(bpy)3]2+ emission and superscript T denotes TINA emission. Buffer and experimental conditions are the same as for Figure SI 2.

13

Figure SI 5. Stern-Volmer plots for TINA D3 (1.0 μM) monomer (a) and excimer (b) quenching by [Ru(bpy)3]2+ at 25 ˚C and 10 ˚C. λex = 375 nm. Buffer conditions are the same as for Figure SI 2.

14

9. K4Fe(CN)6 – [Ru(bpy)3]2+

Fluorescence Quenching Studies

We evaluated how strongly the [Ru(bpy)3]2+ complex interacts with the DNA duplexes by

how efficiently it is protected from quenching by [Fe(CN)6]4- by the method outlined in our

previous paper.15 Initial trials using methyl viologen (MV+) as a quencher were unsuccessful

in the presence of DNA. Free [Ru(bpy)3]2+ and ZnTMpyP4 were quenched by free MV+ in

solution, but in the presence of DNA no appreciable quenching was seen for any of the

duplexes. MV+ has been well documented to interact with DNA,16, 17 possibly competing

with chromophores of interest for DNA binding sites, hence we decided to use K4Fe(CN)6

which does not interfere with DNA.

(c)

Figure SI 6 Plots of relative emission intensity versus stDNA: [Ru(bpy)3]2+ ratio (a) and relative emission intensity versus D1: [Ru(bpy)3]2+ ratio (b). [Ru(bpy)3]2+] = 1.0 µM. Buffer and experimental conditions are the same as for Figure SI 3. (c) Stern-Volmer plots of quenching of [Ru(bpy)3]2+ fluorescence with increasing concentration of a quencher, [Fe(CN)6]4- in the absence and presence of DNAs. [Ru(bpy)3]2+] = 1 µM, DNA: [Ru(bpy)3]2+ = 40:1. Conditions: 25 °C, pH = 7.0, 10 mM sodium phosphate buffer, 0.1 mM EDTA, 50 mM NaCl.

15

Titration of 1 µM [Ru(bpy)3]2+ with stDNA resulted in an emission enhancement of up to

around 1.50, more than that for short stranded D1 (≈ 1.04), Figure SI 6. TINA-containing

short stranded duplexes D2, D3 and D4 caused emission enhancements higher than D1 but

smaller that stDNA and the values were 1.22, 1.41 and 1.36, respectively.

As shown in Figure SI 6c, in the absence of DNA, [Ru(bpy)3]2+ was efficiently quenched by

[Fe(CN)6]4-, resulting in a linear Stern-Volmer plot (slope 2.98, correlation coefficient 0.998,

Table SI 5). In the presence of long stDNA, the slope of the plot is markedly decreased (slope

0.32, correlation coefficient 0.996). This is in contrast to short unmodified D1 (slope 2.61,

correlation coefficient 0.990) which exhibited only a marginal decrease. The presence of the

TINA in duplexes D2-D4 caused a significant decrease in the slopes (Table SI 5), approaching

that of stDNA which nearly equal to zero.

Table SI 5 Slopes of plots on Figure SI 5c representing quenching efficiency of [Ru(bpy)3]2+ fluorescence by [Fe(CN)6]4- in the

presence of DNA duplexes

Duplex Slope R2

value

No DNA 2.98 0.998

D1 2.61 0.990

D2 1.33 0.983

D3 0.96 0.995

D4 1.06 0.994

stDNA 0.32 0.996

The results in Table SI 5 imply that the presence of TINA improves the engagement of

[Ru(bpy)3]2+ within the DNA structure for short-stranded DNA duplexes.

16

10. UV-Vis thermal difference spectra (TDS)

Marked changes in the TDS profiles of the duplexes studied in the presence of [Ru(bpy)3]2+

were quite useful in our analyses (Figure SI 7). [Ru(bpy)3]2+ alone exhibits two prominent

negative TDS peaks around 290 nm and 450 nm. The most significant observation was the

appearance of a negative TDS peak around 450 nm in all duplexes in the presence of

[Ru(bpy)3]2+. There was no sign change on the TDS peak at around 450 nm on adding

[Ru(bpy)3]2+ to all duplexes. There was a decrease in the intensity of the [Ru(bpy)3]2+ peak in

the presence of D2 and D3, but no such change was observed with D4.

Figure SI 7 Changes in thermal difference spectrum of D4 on addition of [Ru(bpy)3]2+ (40 μM). Buffer and experimental conditions are the same as for Figure SI 2.

Addition of one TINA-modified D1 led to the disappearance of the negative [Ru(bpy)3]2+ peak

around 290 nm. On the other hand, in the presence of the doubly TINA-modified duplexes

D2 and D3 the 290 nm negative peak is present, albeit at lower intensities in both cases.

This may suggest that [Ru(bpy)3]2+ participates differently in the duplex folding in the

presence of two TINAs compared to one TINA. The positive DNA peak around 260 nm

increases significantly in intensity in the presence of [Ru(bpy)3]2+ in all duplexes, except D2.

This is partly a result of the presence of two [Ru(bpy)3]2+ positive peaks in the same region,

but this cannot entirely account for the big increase.

Major decreases were observed in the intensities of TINA peaks in the range of 300 - 420 nm

in the presence of [Ru(bpy)3]2+ for D2 and D3 in a low salt concentration, but for D4 there

was an increase (Figure SI 7). These changes suggested that [Ru(bpy)3]2+ interacts with TINA

17

and plays a major role as it is in close proximity to the TINA during duplex folding and

unfolding.

18

11. Circular Dichroism (CD) spectra

In the presence of [Ru(bpy)3]2+, both unmodified and modified duplexes develop a negative

induced CD signal in the [Ru(bpy)3]2+ MLCT band around 450 nm, an increase in the negative

peak around 250 nm, and a new negative peak around 300 nm. The appearance of a

negative induced [Ru(bpy)3]2+ signal, is indicative of complex formation, usually by

intercalation.18 A negative band appearing in the 290 – 300 nm is further evidence of

interaction between the ligand and DNA. Upon binding to the TINA-modified D2 duplex, the

[Ru(bpy)3]2+ complex produced a decrease in the negative ICD magnitude (≈64 %) and a red

shift of 9 nm from 460 nm as compared to unmodified D1.

Figure SI 8 CD spectra of 1.0 µM D1 and D2 in the absence and in the presence of [Ru(bpy)3]2+. Buffer and experimental conditions are the same as for Figure SI 2.

The intensity of the positive TINA signals at 371 and 395 nm decreased upon [Ru(bpy)3]2+

binding. This, just like in the case of porphyrin, indicates that the TINA monomers interact

with [Ru(bpy)3]2+.

19

12. ZnTMpyP4/ TINA – DNAs PUC trials

Table SI 6 A table of unsuccessful PUC experiments attempted using DNAs and ZnTMpyP4. Fluorescence intensity was

measured at 420 nm at different ratios of TINA-modified DNA duplexes and ZnTMpyP4, λex = 565 nm.a

DNA DNA strand concentration

(µM) ZnTMpyP4

concentration (µM)

D2

2.5 5 10 20 40

1 � � � � �

5 � � � � �

10 � � � � �

20 � � � � �

30 � � � � �

40 � � � � �

D3

1 � � � � �

5 � � � � �

10 � � � � �

20 � � � � �

30 � � � � �

40 � � � � �

D4

1 � � � � �

5 � � � � �

10 � � � � �

20 � � � � �

30 � � � � �

40 � � � � �

(a) A tick stands for experiments performed. Conditions: pH = 7.0, 10 mM sodium phosphate buffer, 0.1 mM EDTA, 50 mM NaCl. λex = 500 nm, 25 ̊C. Excitation slit = 4 nm and emission slit = 8 nm. All solutions were carefully degassed with argon for at least 24 hours before measurements. A 400 nm long-pass filter was placed in the fluorimeter excitation beam to prevent second order direct excitation of the donor.

20

13. Characterization of modified DNAs.

13.1 MALDI-TOF spectra

Sequence, X is TINA monomer (calculated and observed values, respectively):

5'-CTCAAGCAAGCT, (3614.4/3615.0)

B1Detector Voltage: 1700VLaser Energy: 93 % 10-Nov-2011SM ON14 UNMODIFIED REF

m/z3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950

%

0

100

SM ON14_AA...AmCit_10-11-2011 55 (0.958) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (53:85) TOF LD+ 8883615.037

3022.069 3602.390

3512.0573484.3873325.049

3312.9213029.4673101.665 3166.4823215.369

3446.1773517.604

3636.164

3651.343

3661.245

3681.0873735.930

3771.401

GAGTTCGTTCGA-5', (3676.4/3676.5),

B3Detector Voltage: 1700VLaser Energy: 96 % 10-Nov-2011SM ON15 UNMODIFIED REF

m/z3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950

%

0

100

SM ON15_AA...AmCit_10-11-2011 17 (0.281) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (1:31) TOF LD+ 5013676.476

3672.576

3670.096

3662.306

3526.9753426.979

3424.9263033.0083365.636

3261.2913118.296 3238.9553319.656

3524.892

3515.177

3568.4243655.585

3677.894

3681.087

3683.571

3686.766

3696.715

3704.186

3796.9433813.899

3817.1503871.903

21

5'-XCTCAAGCAAGCT (4083.1/4086.6)

B9Detector Voltage: 1700VLaser Energy: 100 % 12-Oct-2011B9 SM ON12 F3

m/z3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900

%

0

100

SM ON12F3_AA-Am Ac 52 (0.906) Sm (Mn, 2x3.00); Sb (0,40.00 ); Cm (48:57) TOF LD+ 91.54086.590

3967.8323934.380

3773.916

3616.795 3683.2163879.913

3974.465

4124.813

4555.437

4224.188

4467.8354242.073

4283.3114311.297

4591.429

4612.0604689.037

4790.506

GAGTTCGTTCGAX-5', (4145.1/4148.1)

G5Detector Voltage: 1700VLaser Energy: 92 % 15-Nov-2011SM ON11B SINGLE TINA

m/z3500 3550 3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4100 4150 4200 4250 4300 4350 4400 4450

%

0

100

SM ON11BF2_AA...AmCit_15-11-2011repeat2 16 (0.288) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (1:32) TOF LD+ 2884148.133

4146.627

4145.497

4143.615

4141.732

4139.850

4133.456

4027.7344002.535

3994.399

3993.291

3985.533

3904.723

4132.328

4068.291

4130.824

4123.310

4151.900

4153.408

4167.366

4207.859

4210.136

4213.5534287.139

4216.210 4288.6714290.586

4320.135

4325.1334327.057

4376.054

22

5'-XXCTCAAGCAAGCT, (4551.2/4553.1)

B7Detector Voltage: 1700VLaser Energy: 95 % 10-Nov-2011SM ON12AF2 DOUBLE TINA

m/z4000 4050 4100 4150 4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 4700 4750 4800 4850 4900 4950

%

0

100

SM ON12AF2_AA...AmCit_10-11-2011 95 (1.698) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (1:121) TOF LD+ 5454553.069

4545.574

4540.8434438.173

4237.884

4236.7414146.627

4083.973

4052.640

4218.489

4421.825

4404.341

4395.8074300.169

4244.740 4389.217

4537.297

4527.453

4554.647

4555.437

4564.125

4572.8224690.239

4687.035

4615.636

4691.841

4692.643

4693.845

4701.060

4744.4684826.195

4882.430

4922.152

GAGTTCGTTCGAXX-5', (4613.2/4610.9)

D1Detector Voltage: 1700VLaser Energy: 98 % 10-Nov-2011SM ON13BF3 DOUBLE TINA

m/z4000 4050 4100 4150 4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 4700 4750 4800 4850 4900 4950

%

0

100

SM ON13BF3_AA...AmCit_10-11-2011 3 (0.047) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (1:10) TOF LD+ 1164610.869

4606.899

4482.311

4460.020

4340.5324323.979

4315.907

4079.490

4031.075

4180.2114163.213

4092.200

4227.991

4457.286

4400.849 4456.505

4451.042

4604.914

4597.376

4487.402

4503.087

4591.033

4613.252

4616.828

4620.405

4621.598

4645.086

4747.692

4652.663

4746.886

4726.759

4750.514

4855.4954751.723

4842.0554862.833

4991.3324904.114

23

5'-XTXCTCAAGCAAGCT, (4856.2/4855.1)

G4Detector Voltage: 1700VLaser Energy: 98 % 10-Nov-2011SM ON16AF3 DOUBLE TINA

m/z4000 4100 4200 4300 4400 4500 4600 4700 4800 4900 5000 5100 5200 5300 5400

%

0

100

SM ON16AF3_AA...AmCit_10-11-2011 67 (1.235) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (63:70) TOF LD+ 644855.087

4853.457

4852.234

4851.013

4609.677

4550.7024497.594

4461.5824341.3034289.438

4227.991

4153.408

4432.332

4850.198

4846.940

4838.7994630.748

4771.499

4857.125

4858.348

4992.5714859.164

4860.794

4870.177

4988.027

4945.159

4995.464

4998.772

5011.600

5016.984

5048.934

5320.5575268.208

5159.668

5263.541 5483.9175401.928

GAGTTCGTTCGAXTX-5', (4919.2/4922.6)

H5Detector Voltage: 1700VLaser Energy: 95 % 15-Nov-2011SM ON17A F3 DOUBLE TINA

m/z4500 4550 4600 4650 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450

%

0

100

SM ON17A F3_AA...AmCit_15-11-2011repeat 6 (0.097) Sm (Mn, 2x0.00); Sb (0,40.00 ); Cm (1:25) TOF LD+ 1504922.563

4921.742

4920.101

4918.871

4916.820

4857.533

4617.6234615.636

4602.929

4542.814

4856.3104771.903

4758.1764624.780

4665.438

4864.057

4925.025

4930.363

5058.4974962.037

4978.943

4993.398

5059.745

5062.6595086.825

5145.8145208.097

5389.0395294.563

24

13.2. Denaturing Polyacrylamide gel electrophoresis of modified DNAs.

Purities of ON fractions after reverse-phase HPLC were confirmed by denaturing gel electrophoresis

using 20 % polyacrylamide gel (0.75 mm thickness, 19:1 acrylamide/bisacrylamide ratio) and found

to be more than 90 % pure. Gels were prepared in 1 × TBE buffer (100 mM Tris, 90 mM boric acid,

and 10 mM EDTA) under denaturing conditions (7 M urea). ONs at 80-150 µM concentrations were

loaded onto the gels after pre-incubation at 90 °C for 10 min in the loading buffer (7M urea, loading

dyes (0.25% bromophenol blue, 0.25% xylene cyanol)).

All gel electrophoresis was performed at room temperature. After the electrophoresis, gels were

stained with 5 % Stains-All® in 50 % water/formamide for 5–10 min and then destained in H2O until

complete washing of the dye from the gel background occurred.

In every lane, there are two visible bands after destaining: the upper band is a DNA band and the

lower band is bromophenol blue, which is a faster migrating dye on PAGE.

ON1 – 5'-CTCAAGCAAGCT

ON2 - GAGTTCGTTCGA-5'

ON3 - 5'-XCTCAAGCAAGCT

0N4- GAGTTCGTTCGAX-5'

0N5 - 5'-XXCTCAAGCAAGCT

0N6 - GAGTTCGTTCGAXX-5'

ON7 - 5'-XTXCTCAAGCAAGCT

ON8 - GAGTTCGTTCGAXTX-5'

where X is TINA monomer.

25

Gel 1:

Gel 2:

26

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