Supplementary Information

Praseodymium promotes B-Z transition in self-assembled DNA nanostructures

Madhabi M. Bhanjadeo and Umakanta Subudhi*

DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials

Technology, Bhubaneswar 751 013, India, Academy of Scientific & Innovative Research

(AcSIR) New Delhi 110025, India.

Designing of oligonucleotides for Y-shaped bDNA structure

Oligonucleotides for self-assembly of Y-shaped bDNA were designed from the primer sequences

of β-actin, Catalase and Cu-Zn superoxide dismutase (SOD-1) genes of Rattus norvegicus. The

primers were already known for their selective binding to their respective mRNAs and reported

for gene expression studies (Chattopadhyay et al., 2007; Subudhi and Chainy, 2012). The

sequences of forward and reverse primers of these genes were reoriented and combined to design

individual oligo as a component of bDNA structures.

Scheme 1 Representing (a) the formation of Y-shaped bDNA by self-assembly process and (b) solution-based self-assembly in thermal cycler and characterization through nPAGE.

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2019

For Y-shaped bDNA structures, three different oligonucleotides were used where any two

oligonucleotides shared 50% complementarities with each other as presented in Fig. S1. For

generating various Y-shaped bDNA structures oligonucleotides were designed having 3T or 5T

in the loop as well as variation in the overhang sequences. As a result, 8 different Y-shaped

bDNA structures (bDNA US-17 to US-24) were designed using a combination of 12

oligonucleotides (Table S1). From the below table, we can see that appropriate combination of

oligonucleotides give rise to a different set of Y-shaped bDNA structures from US-17 to US-24.

Oligonucleotides were designed with an internal sequences derived from the reverse and forward

primers of β-actin, and SOD-1. Primarily, each strand contains 48 nucleotides which are

separated by either 3T or 5T in the loop and the 5′ end of the strand has an overhang AATT,

GATC, or AGTC as mentioned in the Table S1. Oligonucleotides were named as a, b, and c

corresponding to the bDNA structure. For example, US-17 has three oligos 17a, 17b, and 17c (55

nt each) and US-18 has three strands 18a, 18b, and 18c (57 nt each). Out of 48 internal

hybridizing sequences of 17a, 24 nucleotides flanking the 5′ end were derived from the β-actin

forward primer whereas the rest 24 internal sequences were complementary to the forward

primer of SOD1. Similarly, oligo 17b has been designed with the reverse primer sequences of β-

actin at the 5′ end, and the rest 24 nt were complementary to the 5′ end of 17a. While the 5′ end

of oligo 17c was designed from the forward primer of SOD-1 and sequences flanking to 3′ end

was complementary to the reverse primer of β-actin.

Likewise, bDNA US-18 is designed where the complementary sequences and overhang

sequences are the same as that of bDNA US-17 but the internal loop length is 5T instead of 3T.

Since internal T loop length play a major role in the flexibility and a determinant factor of the

bent angle between two arms two sets of oligonucleotides were designed with varied T loop

length and ovehnags. bDNA US-17, US-19, US-21, and US-23 were designed to yield Y-shaped

bDNA with 3T internal loop whereas US-18, US-20, US-22, and US-24 were designed with 5T

internal loop keeping the internal complimentary sequences same with different overhangs.

Composition of different oligonucleotides for each bDNA has been presented in Table S1.

Table S1. List of Y-shaped bDNA structures with their individual oligonucleotides.

bDNA structure

Oligo Name

Nucleotide sequences

17a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

17b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-17

17c 5′ AGCT TGA GCA GAA GGC AAG CGG TGA ACC TTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

18a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTTTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

18b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTTTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-18

18c 5′ AGCT TGA GCA GAA GGC AAG CGG TGA ACC TTTTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

19a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

19b 5′ AATT CCT GCT TGC TGA TCC ACA TCT GCT TTT GAA GC T GTA GTC ACG CTC GGT CAG 3′

US-19

19c 5′ GATC TGA GCA GAA GGC AAG CGG TGA ACC TTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

20a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTTTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

20b 5′ AATT CCT GCT TGC TGA TCC ACA TCT GCT TTTTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-20

20c 5′ GATC TGA GCA GAA GGC AAG CGG TGA ACC TTTTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

21a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

21b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-21

21c 5′ GATC TGA GCA GAA GGC AAG CGG TGA ACC TTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

22a 5′ GATC CTG ACC GAG CGT GGC TAC AGC TTC TTTTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

22b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTTTT GAA GCT GTA GCC ACG CTC GGT CAG 3′

US-22

22c 5′ GATC TGA GCA GAA GGC AAG CGG TGA ACC TTTTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

23a 5′ AATT CTG ACC GAG CGT GGC TAC AGC TTC TTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

23b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-23

23c 5′ AGCT TGA GCA GAA GGC AAG CGG TGA ACC TTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

24a 5′ AATT CTG ACC GAG CGT GGC TAC AGC TTC TTTTT GGT TCA CCG CTT GCC TTC TGC TCA 3′

24b 5′ GATC CCT GCT TGC TGA TCC ACA TCT GCT TTTTT GAA GCT GTA GTC ACG CTC GGT CAG 3′

US-24

24c 5′ AGCT TGA GCA GAA GGC AAG CGG TGA ACC TTTTT AGC AGA TGT GGA TCA GCA AGC AGG 3′

Self-assembly of DNA

Oligos were purchased from Integrated DNA Technology (IDT, USA) and directly used in self-

assembly reaction without further fractionation and purification. Self-assembly among the

oligonucleotides was carried out in TAE/Mg2+ self-assembly buffer (1xTAEM) as described

earlier (Nayak and Subudhi, 2014; Nayak et al., 2016). The 1xTAEM buffer consisted of 40 mM

Tris base (pH 8.0), 20 mM acetic acid, 2 mM EDTA and 12.5 mM Mg(Ac)2. In 25 µl reaction,

each oligos were combined in equimolar (50 pmol each) ratio, denatured at 95°C for 9 min and

then cooled to 4°C with ramp rate 0.3°C/sec using a thermal cycler (S1000, Bio-Rad). The self-

assembled bDNA samples were then directly used for characterization in nPAGE and examined

for B-Z transition without further fractionation or purification.

Circular dichroism analysis

CD measurements were carried out in a Chirascan spectrophotometer (Applied Photophysics).

Before starting the experiment nitrogen gas was circulated through the instrument to provide

oxygen-free environment and the purging was continued throughout the experiment. All the

experiments were carried out in an optical cell with path length of 1 mm. CD spectra of all

samples were measured at 20°C with scan speed of 60 nm/min, bandwidth of 1 nm and 0.5 sec

per point. The temperature of the cell was controlled with Industrial Chiller (CW-3000) and

monitored through temperature controller (Quantum Northwest, TC-425). The CD and

absorbance spectrum of 1xTAEM was used as the background and it was subtracted from the

experimental spectra. For sample analysis, 200 µl of 2 µM bDNA were taken in the cuvette and

it was scanned from 200 to 320 nm to obtain the characteristic spectra of B-DNA. Then 0.5 µl of

1 M PrCl3 (prepared in MQ water) was titrated to the former solution consecutively to obtain 2.5

to 10 mM of PrCl3 in the reaction mixture and the respective spectra were recorded.

Simultaneously, the absorbance spectra of bDNA samples were also recorded with and without

PrCl3 in the same Chirascan Spectrophotometer. Data acquisition and analysis was done using

Pro-data Chirascan software. Each smooth CD spectra is an average of three scans subtracted

from 1xTAEM buffer as blank which is also average of three scans.

Fluorescence-based dye binding assay

Fluorescence studies were conducted at 25°C in a quartz cuvette with 1 cm path length using

Hitachi spectrofluorimeter (Model F-2500). To check the EtBr emission, 7 µM of EtBr

(excitation wavelength: 480 nm) alone was taken in Tris buffer and then mixed with 6 µM

bDNA US-23. Then the EtBr-bDNA complex was titrated with different concentrations (2.5, 5.0,

7.5, and 10 mM) of PrCl3. Similar experiments were conducted by taking 10 µM Hoechst 33342

(excitation wavelength: 361 nm) and 7 µM DAPI (excitation wavelength: 358 nm) with 6 µM

bDNA US-21 and then titrated with different concentrations (2.5, 5.0, 7.5, and 10 mM) of PrCl3. Dye binding assay using CD spectroscopy

The DNA binding dyes DAPI (40 µM), Hoechst 33342 (20 µM), and EtBr (120 µM) were used

for the experiment. They did not exhibit any CD signal when run individually, however, these

dyes showed induced peaks in CD spectra when bound to bDNA. First these dyes were

individually added to B-bDNA US-21 and CD spectra were collected. Then dye-bDNA complex

was titrated with different concentrations (2.5 mM to 10 mM) of PrCl3. In another set, bDNA

US-23 was first titrated with PrCl3 (2.5 mM to 10 mM) which caused B-Z transition. Then, DNA

binding dyes were added to Z-bDNA US-23 and both the absorbance and CD were recorded. All

the experiments were conducted at 20°C in 1 mm cuvette. Final spectra are an average of three

acquired spectra which were corrected against assembly buffer as baseline.

Major groove binding assay

Since major groove is missing in the Z-DNA unlike the B-DNA, the formation of Z-DNA can be

easily monitored using CD spectroscopy by assaying the absence of major groove by major

groove binder. Methyl green (MG) is a known major groove binder and after binding to the

major groove of B-DNA, an induced CD signal was obtained at 650 nm. Only MG (25 µM) did

not result any peak in the CD spectra but showed induced peak when bound to DNA. In brief,

MG dye was added to bDNA US-23 and the CD spectra were collected. Then dye-bDNA

complex was titrated with PrCl3 (2.5 mM to 10 mM). On the contrary, bDNA US-23 was first

titrated with PrCl3 (2.5 mM to 10 mM) to initiate the B-Z transition. Then, MG was added to the

Pr-induced Z-bDNA US-23 and recorded the CD spectra along with the absorbance. Final

spectra are an average of three acquired spectra which were corrected against assembly buffer as

baseline.

Analysis of DNA melting curve

Melting curve experiment of both B-and Z-bDNA samples was performed in CD

spectrophotometer to determine the thermal stability of the DNA samples. Sample concentration

and volume remained similar to the earlier measurement. For melting curve analysis of B-bDNA,

the sample was heated from 10 to 90°C with a rate of 0.3°C/min and the data was recorded at 1

nm step with tolerance of 0.02°C. After denaturation, same bDNA samples have been renatured

from 90 to 10°C keeping same set of parameters. Similar pattern was followed to obtain the

melting and annealing curve of lanthanide-induced Z-bDNA. Simultaneously, the absorbance

spectra were also recorded for the melting and annealing curve of B-bDNA and Z-bDNA.

Z-B reverse transition assay using EDTA

EDTA is a known chelator of divalent and trivalent cations. EDTA was applied to the La-

induced Z-DNA, to examine the Z-B transition by withdrawal of Pr3+. Two sets of experiment

were designed, firstly EDTA was added to bDNA US-23 and the CD spectra were collected.

Then EDTA-bDNA mixture was titrated with PrCl3 (2.5 mM to 10 mM). In the second set of

reaction, bDNA US-21 was first titrated with PrCl3 (2.5 mM to 10 mM) which caused B-Z

transition. Then, EDTA was added to the Z-bDNA US-23 and recorded the CD spectra to notice

the Z-B transition. Simultaneously, the absorbance spectra were recorded to observe the change

in absorbance during Z-B transition.

Fig. S1 (a, b) CD spectra of bDNA US-19 and US-20 with PrCl3, (c, d) Absorbance spectra of bDNA US-19 and US-20 with PrCl3.

Fig. S2 (a, b) CD spectra of bDNA US-21 and US-22 with PrCl3, (c, d) Absorbance spectra of bDNA US-21 and US-22 with PrCl3.

Fig. S3 (a, b) Absorbance spectra of bDNA US-17, US-18, US-23 and US-24 with PrCl3.

Fig. S4 (a, b) CD and absorbance spectra of B-bDNA US-23 with DAPI followed by PrCl3 and (c, d) CD and absorbance spectra of Z-bDNA US-23 with DAPI.

Fig. S5 (a, b) CD and absorbance spectra of B-bDNA US-23 with DAPI followed by PrCl3 and (c, d) CD and absorbance spectra of Z-bDNA US-23 with DAPI.

Fig. S6 (a) Absorbance spectra of B-bDNA US-23 with EtBr followed by PrCl3 and (b)

Absorbance spectra of Z-bDNA US-23 with EtBr. (c) Absorbance spectra of B-bDNA US-23 with EDTA followed by PrCl3 and (d) Absorbance spectra of Z-bDNA US-23 with EDTA.

Fig. S7 Fluorescence emission spectra of (a) Hoechst, (b) EtBr, (c) DAPI in presence of bDNA US-23. Different concentrations (2.5, 5.0, 7.5 and 10 mM) of PrCl3 added to the dye-bDNA US-23 complex. (d) Relative fluorescence of different dye-DNA complex and their quenching by PrCl3.

Fig. S8 (a, b) Thermal melting (10 to 90°C) and (c, d) thermal annealing (90 to 10°C) of B-bDNA US-23 by CD spectroscopy.

Fig. S9 (a, b) Thermal melting (10 to 90°C) and (c, d) thermal annealing (90 to 10°C) of B-bDNA US-23 by absorbance spectroscopy.

Fig. S10 (a, b) Thermal melting (10 to 90°C) and (c, d) thermal annealing (90 to 10°C) of Z-bDNA US-23 by CD spectroscopy.

Fig. S11 (a, b) Thermal melting (10 to 90°C) and (c, d) thermal annealing (90 to 10°C) of Z-bDNA US-23 by absorbance spectroscopy.